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Hum Vaccin Immunother. 2014 June 1; 10(6): 1688–1699.
Published online 2014 March 18. doi:  10.4161/hv.28517
PMCID: PMC5396244

Cimetidine synergizes with Praziquantel to enhance the immune response of HBV DNA vaccine via activating cytotoxic CD8+ T cell

Abstract

Previously, we have reported that either CIM or PZQ, 2 clinical drugs, could be used to develop as adjuvants on HBV DNA vaccine to elicit both humoral and cellular immune responses. Here, we demonstrate that combinations of CIM and PZQ as adjuvants for a HBV DNA vaccine, could induce much stronger antigen specific CD4+ and CD8+ T cell responses compared either with CIM or PZQ alone. The synergistic effects of CIM plus PZQ to HBV DNA vaccine were observed on a higher IgG2a/IgG1 ratio, an increase of HBsAg-specific CD4+ T cells capable of producing IFN-γ or IL-17A and a robust IFN-γ-, IL-17A-, or TNF-α-producing CD8+ T cells to HBsAg. Most importantly, the antigen-specific CTL response was also elevated significantly, which is critical for the eradication of hepatitis B virus (HBV) infected cells. Using an HBsAg transgenic mouse model, the expression of HBsAg in the hepatic cells was also significantly reduced after immunized with pCD-S2 in the presence of 0.5% CIM and 0.25% PZQ. Further investigations demonstrated that the synergistic effects of combination of CIM and PZQ were dependent on enhanced cytotoxic CD8+ T cells, which was correlated with impaired activities of regulatory T cells. Therefore, combinations of CIM and PZQ have great potential to be used as effective adjuvants on DNA-based vaccinations for the treatment of chronic hepatitis B.

Keywords: Cimetidine, Praziquantel, adjuvant, HBV DNA vaccine, cytotoxic CD8+ T cell

Introduction

Hepatitis B is one of the most prevalent and serious liver diseases, which was caused by the hepatitis B virus (HBV) infection. At present, there are more than 350 million people suffering from chronic hepatitis B in the world and 15–25% of these infected persons are at high risk of developing serious liver damages, including liver cirrhosis, hepatocellular carcinoma, and even death.1,2 Fortunately, we have safe and effective recombinant hepatitis B surface antigen (HBsAg) vaccines to protect newborns and uninfected individuals from HBV infection.3 In addition, the availability of immunomodulators (interferon α, thymosin α1) and potent nucleoside analogs (such as lamivudine, adefovir, entecavir, etc.) treatments mark a new era in the control of chronic hepatitis B.4-6 However, the therapeutic efficacy of IFN-α is quite limited,7 and long-term treatments with nucleoside analogs appears to show increased rates of resistance to antiviral drugs and other severe side effects.6 In particular, the failure of traditional antiviral strategies to eradicate the HBV infected cells highlights the need for novel therapeutic vaccines development.

DNA vaccines encoding a viral protein represent an attractive strategy to fight against various infectious diseases caused by HBV, human immunodeficiency virus (HIV) and influenza virus infection.8-10 In contrast to commercial recombinant HBsAg vaccines, DNA-based vaccination have great advantages in the ability to induce both humoral and cellular immune responses, which is of great important for eliminating virus infected cells.11,12 Whereas, the relatively weak immune responses in human clinical trials have limited its application,13 which attract our attention on developing potent effective adjuvants to improve the therapeutic efficacy of HBV DNA vaccines.

Because of the relative safety and mild side effects, some small chemical molecules have shown promise to be used as an adjuvants to stimulate the immune responses of DNA based vaccination.14-16 Praziquantel (PZQ), an effective anthelmintic drug against all forms of schistosomiasis,17,18 is explored as an adjuvant for HBV DNA vaccines to facilitate virus specific Tc1and Tc17 responses via the decreased expression of TGF-β from T regulatory cells.19,20 In addition, cimetidine (CIM), a drug that is prescribed for the treatment of heartburn and peptic ulcers,21 is a H2-receptor antagonist, which also could be used as an adjuvant for HBV DNA vaccines to increase the secretion pro-inflammatory cytokines and decrease the production of anti-inflammatory cytokine, IL-10.22-24 Interestingly, CIM also could increase the bioavailability of PZQ due to its ability to reduce the enzyme activities of cytochrome P450, which is crucial for the metabolism of PZQ.25-27 These observations prompted us to investigate whether CIM could act synergistically with PZQ to further enhance the immune responses on HBV DNA vaccine.

In this study, we evaluated the adjuvant effects of CIM plus PZQ on humoral and cellular immune responses triggered by immunization with HBV DNA vaccines (pCD-S2) that encodes hepatitis B surface antigen pre-S2 and S regions. Compared with CIM or PZQ alone, combination of CIM and PZQ further impaired the suppressive abilities of Tregs and significantly enhanced HBsAg-specific CTL response, which is mainly mediated by IFN-γ and IL-17A producing CD8+ T cells. In addition, the therapeutic efficacy of DNA based vaccination in the cooperation of CIM plus PZQ was further confirmed in an HBsAg-Tg mouse model. Therefore, combination therapy with HBV DNA vaccine plus 2 or more adjuvants proposed to be potential immunotherapeutic approaches for the treatment of chronic hepatitis B.

Results

HBsAg-specific humoral immune responses

Hepatitis B surface antibodies (anti-HBs) are a key serological marker that can be used to monitor the vaccine-induced humoral immune responses. To evaluate whether combination of CIM and PZQ as adjuvants for DNA vaccine could induce better humoral response than mice immunized with pCD-S2 and either one, WT C57BL/6 mice were immunized with pCD-S2 DNA vaccine in the presence or absence of CIM, PZQ, or both (Table 1), and the immunization schedule was shown in Figure 1A. Seven days after the third immunization, serum IgG, IgG2a, and IgG1 antibodies against HBsAg were measured by quantitative ELISA method. Compared with the pCD-S2 immunized group, the levels of HBsAg-specific IgG were significantly increased in the presence of CIM, PZQ, or both. Although, combination of CIM and PZQ as adjuvants did not further increase the production of total IgG or IgG2a compared with mice treated with either of them (Fig. 1B and C). The ratio of IgG2a/IgG1 was dramatically elevated in the group of pCD-S2 with 0.5% CIM and 0.25% PZQ, suggesting a Th1 biased response (Fig. 1D).

Table thumbnail
Table 1. Immunization groups
figure hvi-10-1688-g1
Figure 1. Effects of CIM plus PZQ as adjuvants on humoral response. Naive mice were immunized (i.m.) with pCD-S2 in the presence or absence of CIM, PZQ, or combination of CIM and different concentration of PZQ (0.25%, 0.5%, and 1.0%). The immunization ...

Combination of CIM and PZQ induced stronger DTH response and T cell proliferation activities

Delayed-type hypersensitivity (DTH) is a simple in vivo assay of cell-mediated immune function.28 To test the effects of CIM plus PZQ on cell-mediated responses to pCD-S2 DNA vaccine, the mice were immunized the same as before and challenged with HBsAg on their footpads on day 7 after the third immunization. The thickness of footpad was measured and quantified at 48 h after the challenge (Fig. 2A). As shown in Figure 2B, mice immunized with pCD-S2 with the combinations of CIM and PZQ induced stronger HBsAg specific DTH response significantly compared with mice treated with pCD-S2 in the presence of either one. Meanwhile, the proliferation capability of antigen specific T cells was also enhanced after immunized with pCD-S2 and the combinations of CIM and PZQ (Fig. 2C). In addition, the cell-mediated immune responses were inhibited when higher concentration of PZQ was used, indicating a dose dependent response (Fig. 2B and C).

figure hvi-10-1688-g2
Figure 2. Combination of CIM and PZQ as adjuvants on cell mediated immunity. (A) WT C57BL/6 mice were immunized 3 times with the plasmid pCD-S2 in the presence or absence of CIM or PZQ, or combination of CIM and different dosages of PZQ. On day ...

Co-administration of CIM and PZQ with pCD-S2 DNA vaccine enhanced HBsAg-specific CD4+ T cell response

To further investigate the synergistic effects of CIM plus PZQ on HBsAg-specific CD4+ T cell responses, single suspension of splenocytes was prepared from mice on day 7 after the third immunization and re-stimulated with 10 μg/mL HBsAg in vitro. Then, intracellular stainings were performed to measure the production of IFN-γ, IL-4, and IL-17A in CD4+ T cells. As shown in Figure 3A and B, IFN-γ-producing CD4+ T cells was significantly increased after immunized with pCD-S2 DNA vaccine in the presence of 0.5% CIM and 0.25% PZQ compared with the mice treated with pCD-S2 plus CIM alone and a higher IL-17A-producing CD4+ T cells was also observed after co-administration of 0.5% CIM and 0.25% PZQ with pCD-S2 DNA vaccine in contrast to the mice treated with pCD-S2 and PZQ (Fig. 3A and D), whereas, the effects on IL-4-producing CD4+ T cells were minimal (Fig. 3A and C). These results demonstrated that the combinations of CIM and PZQ elicited stronger CD4+ T cell mediated immune response triggered by the pCD-S2 DNA vaccinations.

figure hvi-10-1688-g3
Figure 3. Effects of the combination of CIM and PZQ on CD4+ T cell responses. WT mice were immunized 3 times with the plasmid pCD-S2 in the presence or absence of CIM or PZQ, or combination of CIM and PZQ. Single splenocytes were isolated from ...

Combination of CIM and PZQ with pCD-S2 DNA vaccine could stimulate stronger HBsAg-specific CD8+ T cell mediated cytotoxic response

CD8+ T effector cells suggested to have the ability to lyse virus infected hepatocytes, which is important for the efficient control of chronic HBV infection.29 To assess if combinations of CIM and PZQ could augment HBsAg-specific CD8+ T-cell responses to pCD-S2 DNA vaccine, mice were immunized as previously described (Table 1) and their productions of IFN-γ, IL-17A, and TNF-α in CD8+ T cells were examined on day 7 after the third immunization. We observed that the combination of CIM and PZQ synergistically increased the frequency of IFN-γ-producing and TNF-α-producing CD8+ T cells significantly (Fig. 4A, B, and D) compared with the mice immunized with pCD-S2 with either of adjuvants. While, the production of IL-17A in CD8+ T cells was relatively enhanced in contrast to mice treated with pCD-S2 and CIM alone and no significant changes was observed between PZQ alone and the combination of CIM and PZQ (Fig. 4A and C).

figure hvi-10-1688-g4
Figure 4. Effects of the combination of CIM and PZQ on CD8+ T cell responses. Single splenocytes were isolated from mice in different groups on day 7 after the third immunization and stimulated with 10 μg/mL S208–215 in the presence ...

Next, we evaluated if combinations of CIM and PZQ have an effect on HBsAg-specific cytotoxic response using an in vivo CTL method. As shown in Figure 4E and F, the combination of CIM and PZQ synergistically induced stronger cytotoxic response significantly compared with mice immunized with pCD-S2 DNA vaccine in the presence of either of them. Taken together, these results indicated that CIM and PZQ could act synergistically to enhance CD8+ T cell mediated immune response triggered by the immunization of pCD-S2 DNA vaccine.

The cytotoxic response is dependent on IFN-γ- and IL-17A-producing CD8+ T cells

To further determine which subpopulations of T cell was responsible for the augmented CTL response, CD8KO mice were immunized 3 times with pCD-S2 in the combination of 0.5% CIM and 0.25% PZQ. While, the WT mice were treated under the same protocol and injected with anti-CD3 or anti-CD4 neutralizing antibodies, respectively after the final immunization (Fig. 5A). The depletion efficacy was confirmed by cell surface staining before an in vivo CTL assay (Fig. 5B). We found that the CTL activities were completely abolished when the CD3+ or CD8+ T cells were depleted. Lack of CD4+ T cells seemed to have a little impact on the CTL response (Fig. 5C).

figure hvi-10-1688-g5
Figure 5. CD8+ T cells are critical for the elevated antigen specific cytotoxic response. As shown in (A), naïve mice were immunized 3 times the same as previously described and 100 μg specific neutralizing antibodies against CD3, ...

It has been demonstrated that cytokines (such as IFN-γ, IL-17A, and TNF-α) produced by activated CD8+ T cells were critical for their CTL responses. To confirm which cytokine is more essential for the above CD8+ T cells mediated cytotoxic responses, a in vitro CTL assay was performed as described in the section of Materials and Methods and Figure 5D. Compared with the isotype control antibody treated samples, the CTL response was reduced significantly after blocked with IFN-γ or IL-17A specific antibodies separately. Nevertheless, the cytolysis activity was not affected when treated with anti-TNF antibodies (Fig. 5E). These results indicated that the achievement of antigen specific CTL activities by the combination of CIM and PZQ with pCD-S2 DNA vaccine are largely dependent on the activities of IFN-γ and IL-17A producing CD8+ T cells.

Combination of CIM and PZQ with pCD-S2 DNA vaccine decreased the frequency and suppressive function of Treg

Several studies have demonstrated that CD4+CD25+ T regulatory cells (Treg) could suppress the function of effector T cells and contribute to the impaired immune response in chronic HBV infected patients.30,31 In addition, depletion of Tregs with anti-CD25 mAb showed enhanced HBV-specific CD8+ T cell response.32 Here, we evaluated if the number and suppressive activities of Tregs were affected by the combination of CIM and PZQ with pCD-S2 DNA vaccine. As shown in Table 1, mice were divided into 7 groups and immunized 3 times with different immunization regimens. Seven days after the final immunization, single suspension splenocytes were prepared from immunized mice and used to test the frequency of CD4+CD25+Foxp3+ Tregs and the expression of IL-10 and TGF-β in CD4+CD25+ Tregs by intracellular staining assay. The results showed that the combination of CIM and PZQ could not only decreased the percentage of CD4+CD25+Foxp3+ Tregs (Fig. 6A and B), but also impaired the production of IL-10 and TGF-β in CD4+CD25+ T cells, which is important for the inhibitory function of Tregs, when compared with mice treated with pCD-S2 DNA vaccine in the presence of either of them (Fig. 6C and D). Moreover, the additive effects of CIM plus PZQ on Tregs were dependent on the concentration of PZQ (Fig. 6B and C).

figure hvi-10-1688-g6
Figure 6. Effects of the combination of CIM and PZQ as adjuvants on regulatory T cells. WT C57BL/6 mice were immunized 3 times with the plasmid pCD-S2 in the presence or absence of CIM or PZQ, or both of them, respectively. Seven days after the ...

Combination of CIM and PZQ with pCD-S2 DNA vaccine could break immune tolerance in HBsAg-Tg mice

HBsAg-transgenic mice, which were engineered to produce HBV surface antigen, have been used to mimic the features of chronic hepatitis in humans.33 To investigate the therapeutic effects of the combination of CIM and PZQ as adjuvants to the pCD-S2 DNA vaccine, the HBsAg-Tg mice were immunized with pCD-S2 in the presence or absence of 0.5% CIM, 0.5% PZQ, or combination of 0.5% CIM and 0.25% PZQ. Seven days after the third immunization, the livers were isolated from different groups and lymphocyte infiltrations in the livers were examined by H&E staining method. Compared with the mice treated with pCD-S2 in the presence of either CIM or PZQ, we observed much more lymphocytes infiltrated into the livers after the combination of CIM and PZQ as adjuvants (Fig. 7A). The infiltrated lymphocytes seem to be CD8+ T cells as detected with anti-CD8 specific antibodies using immunohistochemical (IHC) staining method (Fig. 7A). Importantly, no obvious liver damages were observed as a normal liver morphology with these infiltrated CD8+ T cells. Significantly, the HBsAg-secretion hepatocytes were reduced after the combination of CIM and PZQ with pCD-S2 DNA vaccinations, which is consistent with the observed level of CD8+ T cell infiltrations (Fig. 7A and B). However, the concentration of serum HBsAg almost had no changes in different groups (Fig. 7C).

figure hvi-10-1688-g7
Figure 7. The therapeutic efficacy of combination of CIM and PZQ with pCD-S2 DNA vaccine in HBsAg-Tg mice. The transgenic mice were immunized with pCD-S2, pCD-S2/0.5%CIM, pCD-S2/0.5%PZQ or pCD-S2/0.5%CIM/0.25%PZQ. Livers from transgenic mice were ...

Serum alanine aminotransferase (sALT) is commonly used as a diagnostic evaluation of liver injury, which is released into the circulation by lysed hepatocytes. To confirm whether the decrease of HBsAg-producing hepatic cells attributed to the cytolytic ability of infiltrated CD8+ T cells, the level of sALT was measured on day 7 after the third immunization. Compared with mice treated with pCD-S2 in the presence of either CIM or PZQ, an elevated level of sALT was observed in mice receiving the pCD-S2 DNA vaccine in the presence of the combined CIM and PZQ (Fig. 7D).

Discussion

HBV infected patients, especially those with chronic hepatitis B, tend to establish a largely tolerogenic environment.34 It has been demonstrated that the immune response of those patients are too weak to control or eliminate the virus infected cells, resulting in persistent infection.35,36 Using an HBsAg-Tg mouse model, we found that immunization of a plasmid pCD-S2 in the cooperation of CIM and PZQ was sufficient to break the immune tolerance in transgenic mice (Fig. 7). The enhanced antiviral responses ascribe to empowered CD8+ T cells, which could induce the destruction of virus-infected cells both in vivo and in vitro (Figs. 4 and and5).5). Further, these cytotoxic CD8+ T cells are also strongly associated with viral clearance in HBV infected humans.37-39

According to their cytokine-secretion profiles, CD8+ T cells can be categorized into distinct subsets, including IFN-γ-producing or TNF-α-producing T cells (Tc1), IL-4-producing T cells (Tc2), and IL-17A-producing T cells (Tc17).40,41 Recent studies have shown that IFN-γ and TNF-α contributed to the antiviral abilities of CD8+ T cells through cytolytic and noncytolytic pathways in the HBV-transgenic mouse model42,43 or other virus infected diseases.44 In addition, the inhibition of viral replication and the specific destruction of virus-infected cells was significantly reduced in IFN-γ knockout mice.19,45 The cytotoxic effect of IFN-γ was thought mainly via directly induction the secretion of Perforin and granzyme B to lyse the target cells, upregulation of Fas expression on hepatocytes and enhanced expression of death related genes or downregulation of anti-apoptotic molecules.46-48 While, Tc17 cells which are responsible for CD8+ T cell mediated CTL response, displayed protective roles against various infectious diseases49 and in the cancer immunotherapy.50 A causal role for Tc17 in the efficient control of chronic HBV infection was also previously exhibited in HBsAg transgenic mouse model by using IL-17A knockout mice51 or adoptively transfer antigen specific Tc17 cells.19 Here we demonstrated that combination of CIM and PZQ as adjuvants for DNA based vaccination could significantly induce the Tc1 and Tc17 cell subsets (Fig. 4), which is critical for the stronger cytotoxic response. In addition, the results from in vitro CTL assay provide strong support for the concept that IFN-γ and IL-17A are mainly responsible for the CD8+ T cell mediated cytotoxic response (Fig. 5). However, the exact mechanisms of CD8+ T cell mediated cytolytic effect still need our further investigation.

T regulatory cells (Tregs) have been described as CD4+CD25+Foxp3+ T cells, which involves the suppression of immune responses and maintenance of peripheral tolerance.52 Growing evidence reported that a higher frequency of CD4+CD25+ regulatory T cells were associate with the weak immune responses in chronic hepatitis B virus infected patients53 and depletion of Tregs could activate the function of HBV-specific CD8+ T cells.32 Moreover, the suppressive activity of Treg was mainly mediated through the secretion of inhibitory cytokines including IL-10 and TGF-β, the granzymes and perforin-dependent cytolysis pathway, the metabolic disruption of the effector T cells, and modulate the maturation or function of dendritic cells.54 The previous data revealed that CIM, a prescribed drug for peptic ulcer, could inhibit the production of anti-inflammatory cytokine IL-10 via modulation the interaction of histamine with H2 receptors.55 While, PZQ, another prescribed drug for the treatment of Schistoma japonicum infection, have been reported to impair the function of Treg via inhibition TGF-β/Smad2,3 signaling pathway.56,57 Here we found that the frequency and function of CD4+CD25+ Tregs was significantly impaired in the splenocytes of mice immunized with the plasmid pCD-S2 in the presence of 0.5% CIM and 0.25% PZQ, which is responsible for the elevated adaptive immunity (Fig. 6). In addition, several studies have reported that decreased frequency of Treg also results in significantly higher Th17 cells in HBV-infected patients,58-60 which is consistant with our results (Fig. 3D). While the biological function of these activated Th17 cells should be well characterized in future study.

Previously, we have confirmed that CIM, as an adjuvant, have the ability to induce DC activition and promote the secretion of pro-inflammatory cytokine IL-12 via the suppression of anti-inflammatory cytokine IL-10.24,61 While, adjuvant activity of PZQ was mainly to enhance CD8+ T cell mediated CTL response through inhibition of TGF-β.57 However, in chronic HBV infection and other chronic infectious diseases, both innate and adaptive immunity are simultaneously required for their efficient therapy. The obvious advantages to combine CIM with PZQ are described as follows: (1) the combination of CIM and PZQ could develop much more cost-effective stategies to fight against chronic infectious diseases due to CIM’s ability to increase the bioavailability of PZQ and (2) they could induce better therapeutic effects by complementing each other in the way to impair the function of Tregs and enhance CD8 functions. Importantly, aluminum is still the most common adjuvant used in human and the relative safety of CIM and PZQ make them have great potential to be used as effective adjuvants for the development of therapeutic vaccines against chronic infections.

Materials and Methods

Animals and reagents

Female 6–8 wk old C57BL/6 wild type mice were purchased from the Shanghai SLAC Laboratory Animal Co. LTD. In addition, 12-wk-old transgenic mice designed as Tg (Alb-1HBV)44Bri/J62 were ordered from the animal center of Shanghai Public Health Clinical Center. All mice were kept under specific pathogen-free conditions at the Fudan University and handled according to the animal welfare guidelines for experimental animals.

Cimetidine (Sigma) was initially dissolved in 0.15 M hydrochloric acid and then adjusted to neutral pH with 1M sodium hydroxide and subsequently diluted to 0.5% with phosphate-buffered saline (PBS). Praziquantel was purchased from China North Pharmaceutical Group Corporation and dissolved in ethanol to 6.7% as previously described.19 Subsequently, the dissolved PZQ was diluted into 0.25%, 0.5%, and 1.0% with PBS. The purified HBsAg were purchased from Shanghai Guikang Biotechnology, Ltd. The HBsAg specific CTL epitopes S208–215 (ILSPFLPL; H-2b-restricted) and OVA derived CTL peptide OVA257–264 (SIINFEKL; H-2b-restricted) were synthesized by Scipeptide Biotechnology, Ltd.

Plasmid construction and preparations

The plasmid pcDNA3.1-S2 (pcD-S2) was constructed as previously described63 and stored in our lab. Briefly, coding sequences for HBV surface antigen preS2 and S were inserted into EcoRI/KpnI digested pcDNA3.1 vector (Invitrogen) and the identity of the plasmid was verified by sequencing. Subsequently, plasmid DNA were prepared using the EndoFree Plasmid Maxi kit (Qiagen Inc.) according to the manufacture’s instruction.

Immunization

The C57BL/6 mice were randomly divided into 7 groups (5 mice per group) and immunized intramuscularly with 100 μg pcD-S2 alone, 100 μg pCD-S2 in the presence of CIM or PZQ, or 100 μg pCD-S2 premixed with 0.5% CIM plus different concentrations of PZQ (0.25%, 0.5%, and 1.0%) on days 0, 14, and 28. As shown in Table 1.

T cell proliferation assay

Single splenocytes suspensions were prepared from mice in different groups on day 7 after the third immunization. Two × 105 cells were added to each well in 96-well, flat-bottom culture plates in 100 μL volume and stimulated with HBsAg (10 μg/mL) at 37 °C 5% CO2. And 10 μg/mL BSA (irrelevant antigen) or Anti-CD3 (1 μg/mL) + Anti-CD28 (0.5 μg/mL) was used as positive control. After 48 h stimulation, 20 μL of MTT (5mg/mL) was added to each well and incubated for another 4 h. The plates were centrifuged at 1500 rpm for 5 min, the supernatants were removed carefully by pipetting, and then 150 μL of DMSO (Sigma) was added to each well to dissolve the crystal. The absorbance was measured at 570 nm in the xMark Microplate spectrophotometer (Bio-Rad Laboratories).

Enzyme-linked immunosorbent assays (ELISA)

To detect anti-HBsAg-specific IgG, IgG2a, and IgG1 antibodies, serum samples were obtained from mice in different groups on day 7 after the third immunization and determined by the quantitative ELISA method. The 96-well plates were coated with 2 μg/mL of HBsAg in a coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) at 4 °C overnight in 100 μL volume. Then, the plates were washed 4 times with PBST and blocked with 5% of BSA in PBST for 1 h at 37 °C. After 4 washes, the serial diluted HBsAb standards or serum samples (100 μL/well) from individual mice were incubated for 1 h at 37 °C. After 4 washes, 1:5000 diluted horseradish peroxidase conjugated goat anti-mouse IgG, IgG2a, and IgG1 antibodies was added to corresponding wells and the plates were incubated at 37 °C for another 1 h. Then, after 8 washes, 100 μL of TMB dissolved in 0.025 M phosphate-citrate buffer was added to each well and the reaction was stopped by the addition of 50 μL of 2 M H2SO4. The optical density (OD) at 450/620nm was then measured in the xMark Microplate spectrophotometer (Bio-Rad Laboratories). The anti-HBsAb titers were expressed in milli international units per mL (mIU/mL) and determined by comparison with a standard curve generated from measurement of the HBsAb standards from Beijing Kinghaw Biological Pharmacy Enterprise Co. Ltd. All serum samples were determined in triplicates.

In vivo depletion of CD3- and CD4- specific T Cells

Specific lymphocyte subsets were depleted by intraperitoneal injection of specific monoclonal antibodies. Briefly, C57BL/6 mice were injected intraperitoneally with anti-mouse CD3 (17A2), anti-mouse CD4 (GK1.5), or rat IgG2a κ chain isotype control antibody (eBR2a) in 200 μL phosphate-buffered saline (PBS) on days 0, 4, and 6 after the third immunization of pcD-S2 plus CIM and PZQ. Depletion efficiency was measured by cell surface staining with APC-Cy7 labeled anti-mouse CD3 mAb (17A2), APC labeled anti-mouse CD8 mAb (53–6.7) and FITC labeled anti-mouse CD4 mAb (GK1.5), then the stained cells were detected on LSR Fortessa (BD Biosciences). The depletion efficiencies were greater than 90%. All antibody used were purchased from eBioscience.

Delayed-type hypersensitivity assay

All mice in different groups were injected with HBsAg (10 μg/50 μL) in the right footpad as test and saline solution in the left footpad as negative control on day 7 after the third immunization. After 48 h, the thicknesses of footpads were measured with a micrometer and calculated by the following formula: Footpad Thickness = thickness of right footpad − thickness of left footpad.

Intracellular cytokines staining

Single splenocyte suspensions were prepared from mice on day 7 after the third immunization and 1 × 106 cells were added to 96-well, round-bottom culture plates in 100 μL volume. For detection cytokines secreted from CD4+ or CD8+ T cells, splenocytes were stimulated with HBsAg (10 μg/mL) or S208–215 (10 μg/mL) for 24 h at 37 °C, 5% CO2 in the presence of anti-CD28 (0.5 ug/mL). The phorbol 12-myristate 13-acetate (PMA) and ionomycin were used as a positive control. In the last 6 h of incubation, protein transport inhibitor containing 3 μg/mL Brefeldin A (BD Biosciences) was added into the culture medium. After stimulation, intracellular cytokines staining was performed as previously described. Briefly, cells were pre-stained with surface markers (such as CD3, CD4, or CD8) specific monoclonal antibodies at room temperature for 20 min. After washes, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% saponin (Sigma-Aldrich). For immunostaining of cytoplasmic IFN-γ, IL-4, IL-17A, and TNF-α, the appropriate fluorescent-labeled anti-mouse monoclonal antibodies were added and stained on ice for 3 h. Finally, cells were washed with PBS and acquired on BD Fortessa flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star). All fluorescent-labeled antibodies used were purchased from eBioscience.

Cytotoxic lysis assay

For in vivo CTL, splenocytes from syngeneic naive C57BL/6 mice were divided into 2 parts. One part was pulsed with 10−6 M HBsAg-derived peptides S208–215 and labeled with 20 μM of CFSE (defined as CFSEhigh target cells). The other part was pulsed with 10−6 M OVA-derived peptides OVA257–264 and labeled with 1μM of CFSE (defined as CFSElow cells) as a non-target control. Cells from the 2 parts were mixed in a 1:1 ratio and injected into immunized recipient mice at 2 × 107 total cells per mouse via the tail vein on day 7 after the final immunization. Eight hours later, splenocytes were isolated from the recipients and their differential CFSE fluorescent intensities were measured with a BD FACS LSR Fortessa (BD Biosciences). Specific lysis was calculated using the following formula: Percentage specific lysis = (1 − [ratio unprimed/ratio primed] × 100), where ratio = percentage CFSElow/percentage CFSEhigh.

For in vitro CTL, the splenocytes were prepared from mice on day 7 after the third immunization and the effector CD8+ T cells were purified with MagCellect mouse CD8+ T cell isolation kit (R&D Systems) according to the manufacturer’s instructions. Subsequently, the collagenase digested hepatic cells from untreated HBsAg-Tg mice were labeled with 20 μM of CFSE and used as target cells. After washing with RPMI1640, the purified effector CD8+ T cells were co-cultured with target cells (E:T = 10:1) in the presence of different neutralizing antibodies against IFN-γ (XMG1.2), IL-17A (eBio17CK15A5), and TNF-α (MP6-XT3) or isotype control in the 96-well plate at 37 °C, 5% CO2. After 48 h co-culture, cells were washed and measured on BD Fortessa flow cytometer (BD Biosciences). Decreases in the frequency and intensity of CFSE labeled cells were used to calculate the cytolysis ability. All neutralizing antibodies were purchased from eBioscience.

Histology and immunohistochemistry (IHC)

Seven days after the final immunization, the livers from the immunized HBsAg-Tg mice were collected and fixed in 4% neutral buffered paraformaldehyde (Sigma-Aldrich). Following fixation, the liver was trimmed, embedded in paraffin, cut into about 4 µm sections, and stained with hematoxylin-eosin (H&E) for histopathological evaluations.

For IHC staining, unstained slides were prepared from formalin-fixed, paraffin-embedded tissues. Antigen retrieval using EDTA was conducted before incubation with anti-mouse CD8 mAb and anti-HBsAg mAb. Secondary antibody incubation was performed with horseradish peroxidase-conjugated goat anti-mouse IgG. The images were captured with an EVOS XL light microscope (Advance microscopy group) to determine the histological changes.

Statistical analysis

Statistics were performed using GraphPad Prism Software 6.0 (GraphPad) and presented as mean ± SEM. An unpaired student’s t test analysis was used for all data analysis. *P < 0.05 indicates statistical significance.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported in part by the National High-Tech 863 Project of China (2012AA02A407 and 2012AA02A406) and Department of Scientific and Technology of Shanghai Municipal Government (2013QLG009) to BW. We thank Dr Jane Q.L. Yu and Mr Zhonghuai He for their assistance in this work.

Glossary

Abbreviations:

CIM
cimetidine
PZQ
Praziquantel
HBV
hepatitis B virus
HBsAg
hepatitis B virus surface antigen
Treg
T regulatory cell
DTH
delayed-type hypersensitivity
CTL
cytotoxic T lymphocytes
TNF-α
tumor necrosis factor-α

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