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Hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) combine CpG oligodeoxynucletides as a novel therapeutic vaccine for chronic hepatitis B infection Jianqiang Li a,∗,1 , Jun Ge a,1 , Sulin Ren a,1 , Tong Zhou a , Ying Sun a , Honglin Sun a , Yue Gu a , Hongying Huang a , Zhenxing Xu a , Xiaoxiao Chen a , Xiaowei Xu a,b a b

Jiangsu Theravac Bio-pharmaceutical CO., Ltd, Nanjing 210042, China State Key Laboratory of Nature Medicines, China Pharmaceutical University, Nanjing 210009, China

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Article history: Available online xxx Keywords: HBV CpG Cellular immune response Protein vaccine Immune tolerance

a b s t r a c t Hepatitis B virus infection is a non-cytopathic hepatotropic virus which can lead to chronic liver disease and hepatocellular carcinoma. Traditional therapies fail to provide sustained control of viral replication and liver damage in most patients. As an alternative strategy, immunotherapeutic approaches have shown promising efficacy in the treatment of chronic hepatitis B patients. Here, we investigated the efficacy of a novel therapeutic vaccine formulation consisting of two HBV antigens, HBsAg and HBcAg, and CpG adjuvant. This vaccine formulation elicits forceful humoral responses directed against HBsAg/HBcAg, and promotes a Th1/Th2 balance response against HBsAg and a Th1-biased response against HBcAg in both C57BL/6 and HBV transgenic mice. Vigorous cellular immune response was also detected in HBV transgenic mice, for a significantly higher number of HBs/HBc-specific IFN-␥ secreting CD4+ and CD8+ T cells was generated. Moreover, vaccinated mice elicited significantly intense in vivo CTL attack, reduced serum HBsAg level without causing liver damage in HBV transgenic mice. In summary, this study demonstrates a novel therapeutic vaccine with the potential to elicit vigorous humoral and cellular response, overcoming tolerance in HBV transgenic mice. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Hepatitis B virus (HBV) infection is the most common cause of liver disease worldwide, and can be transmitted parenterally, sexually and perinatally [1]. Only 5–10% HBV infected adults become persistently infected and chronic carriers, while neonatally transmitted HBV infection is rarely cleared and up to 90% of infected children become chronically infected [2]. It is estimated that more than a third of the world’s population has been infected with HBV, 350 million of whom are chronically infected and at risk of liver diseases, such as liver cirrhosis and cancer [3]. Despite the availability of effective prophylactic vaccine for a long time, up to 10% population is still unable to response with an adequate antibody level to hepatitis B surface antigen [4]. Antiviral therapies currently used in clinical treatment of chronic hepatitis B infection include peg-interferon and standard ␣-interferon,

∗ Corresponding author. Tel.: +86 25 85560000x3052. E-mail addresses: [email protected], [email protected] (J. Li). 1 These authors contributed equally to the work.

nucleoside (lamivudine, entecavir, tenofovir and telbivudine) and nucleotide (adefovir) analogues, both of these have potent activity against HBV [5]. However, nucleotide or nucleoside analogues lead to frequent relapse in the short-term treatment and resistant viral variants in the long-term treatment [6]. The apparent side-effect profile of interferon limits its long-term use. Moreover, so far combination therapy with nucleoside analogue and peg-interferon or two nucleoside analogues does not show a clear benefit as compared with monotherapy [7,8]. All of these treatments lead to viral rebound once the drug discontinued and cannot eradicate the virus completely. Therefore, more effective therapeutic approaches are urgently needed. In this regard, continuous efforts have focused on exploring specific immunotherapeutic strategies as possible alternatives to antiviral drugs and ␣-interferon in chronically infected patients. Among these, recombinant protein based vaccines including HBsAg-containing particles alone or HBsAg combines Th1 promoting adjuvants or HBsAg combines antiviral drugs [9–11], DNA vaccines encoding HBV antigens were designed to specifically stimulate HBV-specific T-cells responses [12–14]. Other strategies being explored include peptides based vaccines and antigen-antibody

http://dx.doi.org/10.1016/j.vaccine.2015.03.079 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Li J, et al. Hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) combine CpG oligodeoxynucletides as a novel therapeutic vaccine for chronic hepatitis B infection. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.079

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complex based vaccines (HBsAg complexed with human anti-HBs) [15,16]. Though some of these strategies have been demonstrated to have the ability to promote the seroconversion of HBeAg and generate partial HBV-specific cytotoxic T lymphocyte (CTL) responses, none of these could achieve full control over HBV. The immune determinants of successful clearance of HBV are still not fully elucidated, previous studies have shown both humoral and cellular immune responses are thought to be important [17]. HBsAg alone is a poor immunogen and requires an adjuvant to enhance its efficacy. Conventional alum adjuvant is typically associated with Th2-biased immune response rather than with cell-mediated immunity [18]. It has been reported that unmethylated cytosine-guanine dinucleotide (CpG) could induce strong Th1 cytokines dominated by IL-12 and IFN-␥ with little secretion of Th2 cytokines, which provide additional T-help for both humoral and cellular immune responses [19]. HBcAg activates B cells to work efficiently as primary antigen presentation cells (APCs) and has a synergistic effect on antibody production and cellular responses when co-administered with HBsAg [20–22]. Besides, it has been reported that patients with Chronic Hepatitis B who could repress HBV replication have significantly higher levels of HBcAg specific CTL(cytotoxic T lymphocyte) compared with those who fail to repress HBV replication [23]. In this study, we devised a novel vaccine formulation consisted of HBsAg, HBcAg and CpG oligodeoxynucletides, and evaluated its efficacy on humoral and cellular immune responses against HBV.

1 h at 37 ◦ C. Sera were diluted in PBST containing 2% skim milk and incubated at 37 ◦ C for 1 h. After washing, the bounded antibodies were detected with IgG (Sigma-Aldrich), IgG1 and IgG2a (Southern Biotech). Antibody titers were determined for each sample as end-point dilution with an arbitrary cutoff OD value of 0.100. Antibody titers of each group were transformed logarithmically and expressed as geometric means ± standard errors (GMT ± SE). For HBsAg quantification, HBV transgenic mice sera were collected and measured using commercially available kit from Kehua Bio-engineering Co., Ltd (Shanghai, China). Protective antiHBs antibody was detected with ARCHITECT5 Anti-HBs Reagent Kit (Abbott).

2. Materials and methods

1 × 106 splenocytes per well were plated on 96-well plates for ELISpot assays for IFN-␥ or IL-4 release using commercially available kit (BD Biosciences). Cells were stimulated with 1 ␮g/ml HBsAg or HBcAg peptides pool for 24 h at 37 ◦ C, 5% CO2 . After washing and counter staining, the bound IFN-␥/IL-4 were detected with biotinylated anti-mouse IFN-␥/IL-4 antibody and HRP-conjugated streptavidin. Spots were counted on a CTL ImmunoSpot analyzer (Cellular Technology Limited).

2.1. Antigens, adjuvant and formulation Recombinant HBsAg particles of subtype adw and adr used in this research were obtained from ProSpec (Rehovot, Israel). Recombinant HBcAg particles containing 183 amino acids were produced in Escherichia coli and purified roughly according to the method described by Birnbaum et al. [24]. Fully phosphorothioate-modified CpG-dinucleotides (CpG-ODNs) were synthesized by Ribo Life Science (Suzhou, China) with purity higher than 99% and the final products contained no detectable levels of endotoxin. The sequence of CpG-ODNs used in the present study was according to the patent described by Xu et al. (China patent No. CN101492672). Three components were mixed and placed on ice for 30 min before immunization. 2.2. Mice immunization C57BL/6 mice (SPF grad) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). C57BL/6 HBV transgenic mice (SPF grad) carrying full-length HBV genome (adr subtype, C genotype) were purchased from Shanghai Biomodel (Shanghai, China). C57BL/6 mice were immunized i.m. three times in 100 ␮l of total volume with indicated formulations containing 1 ␮g adw subtype HBsAg, 1 ␮g HBcAg and 2 ␮g CpG, respectively, if not otherwise stated. HBV transgenic mice were immunized i.m. six times in 100 ␮l of total volume with indicated formulations containing 10 ␮g adw subtype HBsAg, 10 ␮g HBcAg and 2 ␮g CpG, respectively. Both C57BL/6 and HBV transgenic mice were immunized two weeks apart. Sera and spleens were collected day 10 after last immunization for humoral and cellular immune responses analysis. 2.3. ELISA 96 wells ELISA plates (Nunc) were coated overnight at 4 ◦ C with 25 ng/well adw subtype HBsAg or 50 ng/well HBcAg, if not otherwise stated. Plates were blocked with 5% skim milk in PBST for

2.4. Peptides pool preparation Peptides pool of HBsAg and HBcAg were synthesized by Sangon Biotech (Shanghai, China) according to the sequence of HBV adw subtype. Each peptide contains 15 amino acids, and the C-terminal and N-terminal of adjacent peptides have 11 amino acids overlap. All these peptides were dissolved in double distilled water or DMSO at the concentration of 10 ␮g/ml. Peptides pool was produced by mixing each peptide with same volume. Finally, 1 ␮g/ml HBsAg peptides pool and 1 ␮g/ml HBcAg peptides pool were used to perform the indicated experiments. 2.5. ELISpot

2.6. Intracellular cytokine staining Freshly prepared splenocytes (4–6 × 106 cells per well) were incubated with 1 ␮g/ml HBcAg, 1 ␮g/ml HBsAg peptide pool and RPMI-1640 medium supplemented with 10%FBS, 1% PSG and 0.1% ␤-mercaptoethanol (GIBCO) as control for 6 h at 37 ◦ C, 5% CO2 , respectively. Golgistop (BD Biosciences) was added to each well to prevent the excretion of cytokines. After stimulation, cells were washed and stained with FITC-conjugated anti-mouse CD8 antibody and PE-conjugated anti-mouse CD4 antibody (BD Biosciences). After fixation and permeabilization, cells were then stained with APC-conjugated anti-mouse IFN-␥ antibody (BD Biosciences). Cells were collected by flow cytometry (BD Biosciences) to calculate the number of responding cells per 105 cells and the data were analyzed by FlowJo software (Tree Star). Background levels of cytokine excretion from control group were deducted from the corresponding results. 2.7. In vivo cytotoxic T lymphocyte (CTL) assay The in vivo cytotoxicity assay was conducted essentially as described previously [25]. Splenocytes from naïve C57BL/6 were labeled with 4 ␮M and 0.4 ␮M CFSE (Molecular Probes). The CFSEhigh (4 ␮M CFSE) were incubated with 1 ␮g/ml HBsAg and 1 ␮g/ml HBcAg peptide pool for 4 h at 37 ◦ C, 5% CO2 , respectively. Both CFSEhigh and CFSElow were washed and mixed at a 1:1 ratio and then transferred retro-orbitally into the immunized recipient mice (2 × 106 cells per mouse in 100 ␮l volume). After overnight (15∼17 h) killing, splenocytes were isolated from the recipient mice and analyzed using flow cytometry (BD Biosciences) for target cells

Please cite this article in press as: Li J, et al. Hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) combine CpG oligodeoxynucletides as a novel therapeutic vaccine for chronic hepatitis B infection. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.079

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Fig. 1. HBsAg/HBcAg/CpG vaccine generates vigorous HBV-specific humoral immune responses in C57BL/6 mice. Sera were collected 10 days after last injection and ELISA were performed to measure the HBV-specific antibody. Groups of C57BL/6 (n = 10) were immunized i.m. three times with indicated formulations, respectively. (A) Anti-HBs antibody titers, (B) IgG subclass patterns of anti-HBs, and (C) IgG subclass patterns of anti-HBc in C57BL/6 mice. Data of each group were transformed logarithmically and expressed as geometric means ± standard errors (GMT ± SE). * p < 0.05, ** p < 0.01 and *** p < 0.001 was determined by ANOVA, followed by Dunnett’s t-test. Titer = 1 means the minimum dilution assay in which no positive signal was detected.

clearance. Gated on CFSE cells, the percentage of antigen-specific killing was determined as follows:



 1−



%CFSEhigh in immunized mice/%CFSElow in immunized mice





%CFSEhigh in naive mice/%CFSElow in naive mice

2.8. Liver histology HBV transgenic mice livers were fixed in 4% buffered formalin for 24 h and embedded in paraffin. Four ␮m sections were treated according to the conventional hematoxylin and eosin (HE) staining methods [26]. Masson’s trichrome staining was performed to detect fibrillar collagen in the tissues according to the methods described by Tsai et al. [27]. Stained tissue slices were microscopically examined at 200× magnification. Five vision fields per mouse were chosen randomly and analyzed by the medical image software program (Image-Pro Plus, Medical Cybernetics, USA). 2.9. Statistical analysis For statistical analysis to compare the immune response between groups of experimental mice, ANOVA and Dunnett’s t-test was performed using Prism 5 (GraphPad Software). Results with a p-value