Recombinant hepatitis B core antigen carrying preS1 epitopes induce immune response against chronic HBV infection

Vaccine 22 (2004) 439–446

Recombinant hepatitis B core antigen carrying preS1 epitopes induce immune response against chronic HBV infection Xinchun Chen1 , Meizhong Li, Xiaohua Le, Weimin Ma, Boping Zhou∗ Shenzhen Institute of Hepatology, 2019 Buxin Road, Shenzhen 518020, PR China Received 16 December 2002; received in revised form 25 July 2003; accepted 28 July 2003

Abstract Many studies have provided evidence that core antigen of hepatitis B virus (HBcAg) is extremely immunogenic, HBcAg can be function as both a T-cell-dependent antigen and a T-cell-independent antigen, and thus may be a promising candidate for therapeutic vaccine for control of chronic HBV infection. HBcAg is also an effective carrier for heterologous peptide epitopes. The preS1 is a surface protein of HBV and is immunogenic at the T and B cell level. The amino acid sequence 21–47 of preS1 is crucial for HBV binding to human hepatocytes as well as to PBMC and haematopoietic cell lines of the B cell lineage. Here we expressed a chimeric protein named HBVCS1, created by fusing the preS1 sequence 3–55 to the carboxyl terminus of the truncated HBcAg sequence 1–155 in E. coli. Analysis of its antigenicity and immunogenicity revealed that both HBc and preS1 epitopes are surface accessible, and that fusion of preS1 did not affect the HBc antigenicity and immunogenicity of the truncated HBc sequence. HBVCS1 induced strong anti-HBc and moderate anti-preS1 immune responses as well specific T-cell response in Balb/c mice. HBVCS1 vaccination reduced of the titer of HBsAg and HBV DNA in sera of HBV-Tg mice. These results indicate that HBVCS1 may have potential as a therapeutic vaccine for treatment of HBV chronic infection. © 2003 Elsevier Ltd. All rights reserved. Keywords: Therapeutic vaccine; HBcAg; preS1

1. Introduction It is estimated that there are 400 million carriers of hepatitis B virus (HBV) by the year 2000 according to the WHO. Among these, approximately 5–10% of adults and 80–90% of children become chronic carriers of HBV. The long term consequence of chronic carriage are cirrhosis, liver failure and hepatocellular carcinoma [1,2]. Currently, the most effective drugs used for treatment of chronic hepatitis B (CHB) are interferon (IFN) and nucleoside analogues, lamivudine. Interferon-␣ achieves a short-term outcomes of around 20–30% loss of HBeAg. The efficacy is even lower in Chinese patients, who are immunotolerant to HBV because of acquisition of the disease during early childhood, than in white patients. Lamivudine, nucleoside analogues, is able to reduce HBV DNA to undetectable levels. However, cessation of treatment usually leads to a rapid relapse of ∗ Corresponding author. Tel.: +86-755-25636998; fax: +86-755-25604034. E-mail address: [email protected] (B. Zhou). 1 Present address: Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724, USA.

0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2003.07.014

disease, and long-term treatment often results in the selection of lamivudine resistant viral variants due to mutation of YMDD motif of the HBV polymerase gene [2]. These outcomes emphasize the need for novel therapeutic approaches. Specific immunotherapeutic strategies have been proposed as possible alternatives to the use of IFN or antiviral drugs to break the non-responsiveness of T-cell immunity in chronically infected patients [3]. Among these, specific vaccine therapies with recombinant protein vaccines [4,5] and gene vaccine [6] and peptide vaccine [7,8] have been studied with animal models or in human clinical trials. HBV core antigen (HBcAg) has been demonstrated to be the immunodominant antigen at the Th cell and CTL level in patients with self-limited HBV infection and might, therefore, be relevant for virus control [8–11]. Recombinant HBcAg can induce strong HBV core (HBc) specific Th cell and antibody response in mice reconstituted with PBMC from patient with chronic HBV infection [12]. HBcAg is also an effective carrier for heterologous epitopes, including preS1, and HBcAg enhances the immunogenicity of the heterologous epitopes [13,14]. The preS1, one of the surface proteins of HBV, because of its immunogenicity, has been used to develop a third generation HBV vaccine named

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Hepacare® [15]. The preS1 is immunogenic at the T and B cell level, and a preS1 specific T-cell response can bypass non-responsiveness to the preS2 and S region of HBsAg [16]. In humans, amino acids (aa) 21–28 of preS1 were identified as a dominant T-cell recognition site and aa 12–32 and aa 32–53 were B cell determinants [17–19]. In addition, the aa 21–47 of preS1 is crucial for HBV binding to human hepatocytes, PBMCs and haematopoietic cell lines of the B cell lineage [20,21]. Antibody directed against this epitope could protect chimpanzees from HBV infection [22]. Based on this knowledge, we created the experimental vaccine HBVCS1, a fusion protein comprised of amino acids 3–55 of preS1 added at the C-terminus of HBV core truncated protein (amino acids 1–155). We evaluated HBVCS1 with respect to its immunogenicity and virological efficiency on HBV chronic infection in na¨ıve Balb/c mice and HBV transgenic (Tg) mice. 2. Materials and methods 2.1. Amplification of the HBV core gene, preS1 gene and fusion gene CS1 The fragment of HBV core gene coding for aa 1–155 and of preS1 coding for aa 3–55 were amplified from serum of a patient with chronic HBV infection by PCR. The primers were designed according to the sequence of the HBV adr strain (Genbank, M38636). The detailed sequences of the primers are as followings, C1 and C2 are for the HBV core gene, while S1 and S2 are for the preS1 gene. C1: 5 -GCCATGGACATTGACCCG-3 (1898nt–1914nt). C2: 5 -GGACCCGCCTCGTCGTCT-3 (2346nt–2363nt). S1: 5 -GGGTCCGGTTGGTCTTCCAAACC-3 (2852nt–2868nt). S2: 5 -GGGATCCTATGGCCAGTGATCCTTG-3 (2988nt–3106nt). The restriction sites of NcoI and BamHI (underline) were introduced into the C1 and S2 sequences, respectively for gene cloning purpose. To allow junction of the HBV core and preS1, the codon for aa 154 of HBV core in C2 was changed to GGG (gly) from AGG (arg), addition of GGG (gly) was introduced to the 5 end of S1 to make it complementary to C2. As a first step in the amplification of HBV gene, HBV template DNA was extracted from the serum of a patient with HBV chronic infection (HBsAg, HBcAg and anti-HBc were positive). Serum was incubated in lysis buffer containing NP-40 for 1 h at 62 ◦ C, followed by routinely phenol–chloroform extraction. First round PCR was performed to amplify HBV core and preS1 fragments, using primers C1/C2 and S1/S2, respectively. The PCR product of HBV core is 465 bp and that of preS1 gene is 159 bp. Second round PCR was used to join the HBV core and preS1 fragments. In a 500 ␮l eppendoff tube, each of following

was added, 74 ␮l of ddH2 O, 10 ␮l of 0.2 mM dNTPs, 10 ␮l of 10× PCR buffer, 2 ␮l (6 U) of Taq polymerase, 1 ␮l each of the PCR products of first round amplification of HBV core and preS1, and 100 ␮l mineral oil. The reaction tube was subjected to amplification for six cycles under the conditions of 94 ◦ C, 38 s; 55 ◦ C, 30 s and 72 ◦ C, 30 s. At the end of the sixth cycle, 25 pmol (1 ␮l) of primer C1 and S2 were added to the reaction tube. An additional 30 cycles of amplification were performed to obtain a product of 624 bp, named CS1. The PCR product was subjected to electrophoresis on 1.2% agarose. 2.2. Construction of expression vector pCS1 To construct the expression vector for expression of the fusion gene CS1, plasmid pET-11d (Novagen, USA) was used. The PCR product, CS1, was purified after electrophoresis using a DNA purification kit (Promega, USA) and then subjected to digestion by NcoI and BamHI (Biolabs, USA). Digested CS1 was purified and inserted into pET-11d which had been digested by the same restriction enzyme to construct pCS1. pCS1 was transfected into E. coli BL21 (Novagen, USA). After propagation, pCS1 was extracted using a commercial kit (Promega, USA) and subjected to identification by restriction enzyme and direct sequencing. 2.3. Expression of HBVCS1 in E. coli pCS1-transfected E. coli BL21 cell were first cultured in LB. After propagation, a clone was selected and transferred to LB broth containing 100 ␮g/ml ampenicillin. Cells were incubated at 37 ◦ C with shaking at 225 rpm for 3–4 h until the culture reached an OD of 0.3–0.4. IPTG was then added to the broth at a final concentration of 1 mM to induce the expression of HBV CS1. Culture was continued for 2 h and BL21 cells were harvested by centrifugation. The pellet of BL21 cells was resuspended in lysis buffer containing lysozyme and subjected to ultrasound. The resulting supernatant and sediments were separated by centrifugation at 12,000 rpm, at 4 ◦ C for 10 min and stored at −20 ◦ C. Subsequently, the supernatant and sediment were subjected for SDS-PAGE to analyze of the expression of HBVCS1 protein. HBVCS1 was purified from the supernatant by first size-exclusion chromatography (G3000SW TSK-GEL, TOSOHAAS, Japan) and then ion-exchange chromatography (Poros20HQ, ABI, USA). 2.4. Western blot analysis of the HBVCS1 The lysates of E. coli BL21 cells transfected with pCS1 induced by IPTG at different concentration of 0, 0.5, 0.75 or 1 mM, the supernatant of pCS1-transfected E. coli BL21 cells were subjected to SDS-PAGE, subsequently electroblotted onto nitrocellulose membrane. The membrane was pre-incubated for 1 h with 5% powdered lipid-free milk

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in PBST (PBS containing 0.1% Tween 20) and subsequently hybridized with mouse anti-HBc for overnight at 4 ◦ C. The membrane was washed with PBST, and incubated with rabbit anti-mouse antibody conjugated with HRP (Dako Co., Denmark) for 1 h. Finally, the membrane was developed with ECL reagent (Amersham Co., UK) followed by exposure to X-film. 2.5. Analysis of preS1 antigenicity of HBVCS1 by ELISA Evaluation of the preS1-specific antigenicity of HBVCS1 was performed by ELISA using a commercial kit (Shanghai alfa bio-technique company, China) with some modification. Briefly, a 96-well plate was coated with anti-preS1 mAb, purified HBVCS1 protein at different concentration were added to wells and incubated for 1 h at 37 ◦ C. Subsequently, the plate was washed three times with PBST and then horseradish peroxidase labeled anti-preS1 mAb was added to wells and incubated for 30 min at 37 ◦ C. Then following the protocol of the kit to wash and develop color and determine the results.

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termine the level of anti-preS1. Briefly, preS1 was used as the solid phase, after blocking with PBST supplemented with 10% fetal calf serum; the sera of mice were added. After extensive washing, the bound antibodies were detected with goat anti-mouse IgG labeled with horseradish peroxidase. Antibody titers were determined by the serial end-point dilution method. Sera from non-immunized mice without dilution were used as negative control. OD value of sample ≥2.1 times of that of negative control was considered positive. The subtypes of anti-HBc in Balb/c mice were determined by goat anti-mouse IgG1 and goat anti-mouse IgG2a (Sigma, kindly provided by Dr. Zhang, Sun-Yat-Sen University of Medical Science).The sera from HBV-Tg mice were also used to detection of HBsAg using a commercial ELISA kit (Shangdong 3V Co., China). The titer of sera was also determined by serial end-point dilution method. Anti-HBc, anti-preS1 and HBsAg titers were expressed as group geometric mean ± standard deviation of the mean of individual animal values, which represent the average of duplicate assays. 2.8. T-cell proliferation assays

2.6. Immunization of Balb/c mice and HBV transgenic mice (Tg) Balb/c mice were purchased from the animal facility of Sun-Yat-Sen University of Medical Science. The HBV-Tg mice used in this experiment were provided by infectious disease center of 458 hospital of PLA, PR China, and kept under standard pathogen-free conditions. The HBV-Tg mouse lineage was initially produced on a C57BL/6 background. The transgene in these mice consists of 1.3 copies of the HBV adr complete genome. The HBV-Tg mice express high level of HBsAg in their serum, and a fraction of them (about 40%) have detectable HBV DNA in their serum. Balb/c mice (male and female) and male HBV-Tg mice, 6–8 weeks old, were immunized two times at a 3-week intervals by i.p. Balb/c mice were injected with recombinant HBVCS1 at doses of 0, 1, 2, or 4 ␮g emulsified with an equal volume of complete Freund’s adjuvant (CFA) at the first immunization and with incomplete Freund’s adjuvant (IFA) at the second immunization. HBV-Tg mice were immunized with the same procedure but with doses of HBVCS1 at 0 and 5 ␮g. 2.7. Serologic test At various times before and after immunization, blood was collected from mice by retrobulbar puncture with heparinized glass pipettes and sera recovered by centrifugation were stored at −30 ◦ C for further assay together. Anti-HBc and anti-preS1 antibodies were assayed in serum from Balb/c mice and HBV-Tg mice by specific ELISA. Anti-HBc was assayed using the commercial ELISA kit (Shangdong 3V Co., China). Purified recombinant preS1 (provided by Prof. Guo, Second Military Medical University) was used to de-

Balb/c mice immunized with HBVCS1 or PBS (control) was sacrificed at day 63; and HBV-Tg mice were sacrificed at day 63 or 84, after the first immunization. Splenocytes were harvested from the mouse and co-cultured with or without HBVCS1 (2 ␮g/ml) in RPMI 1640 for 54 h before addition of [3 H] thymidine (1 ␮Ci per well). The cells were harvested 18 h later onto filter strips for determination of [3 H] TdR incorporation. The filters were soaked in 5 ml of solution and the radioactivity measured on ␤ counter (Beckman, USA). The data are presented as counts per minute corrected for background proliferation in the absence of HBVCS1. 2.9. Quantitation of HBV DNA in serum Sera from HBV-Tg mice were subjected to detection of HBV DNA by the fluorescent quantitative PCR method using a commercial PCR kit (AG, USA). 2.10. Statistical methods The significance of the difference between values was assessed with ANOVA and S–N–K test and Pearson correlation analysis using SSPS10.0.

3. Results 3.1. Construction of expression vector pCS1 The CS1 fused DNA sequence, encoding HBV core aa 1–155 joined by aa 3–55 of preS1, which is 624 bp long,

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The DNA sequence of CS1 was found to have a homology of 98.3% aligned with that of HBV adr strain (Genbank, M38636) (data not shown). 3.2. Expression and purification of HBV CS1 in E. coli BL21 A recombinant protein with expected molecular weight of 23 kDa was efficiently expressed in E. coli BL21 as shown by SDS-PAGE (Fig. 1B). The recombinant protein, HBVCS1, was mainly in supernatant of the BL21 lysate. HBVCS1 were purified to approximately 95% purity by chromatography. Western blotting confirmed the specificity of the HBVCS1 (Fig. 1C), which reacted specifically with mAb anti-HBc. In addition, the level of HBVCS1 expression is affected by the concentration of IPTG and the optimal concentration of IPTG is 1 mM (Fig. 1C). The preS1 specific antigenicity of HBVCS1 was demonstrated by ELISA, which showed its reactivity with mAb anti-preS1 at a concentration of 1 ␮g/ml HBVCS1 (data not shown). This also indicated that the fused preS1 epitope is surface accessible, which is a prerequisite for its efficient immunogenicity. 3.3. Strong immune response induced in Balb/c mice immunized with HBVCS1

Fig. 1. Cloning and expression and immunoblot analysis of HBVCS1. (A) Identification of expression vector pCS1 by electrophoresis on 1.2% agarose. Lane M shows DNA molecular weight marker, lane 1 plasmid pCS1, lane 2 pCS1 digested by Stu, lane 3 pCS1 digested by Stu and Pst, lane 4 pCS1 digested by NcoI and BamHI, lane 5 CS1(PCR product). (B) SDS-PAGE analysis of recombinant HBVCS1 protein. MW molecule weight protein markers, lane1 untransfected E. coli BL21 cells, lane 2 pCS1 transformed E. coli BL21 cells not induced by IPTG, lanes 3 and 4 pCS1 transformed E. coli BL21 cells induced by IPTG, lane 5 the sediment of lysate of HBVCS1 expressing E. coli BL21 cells, lane 6 the supernatant of lysate of HBVCS1 expressing E. coli BL21 cells, lane 7 purified HBVCS1. (C) Western blot analysis of HBVCS1 lanes 1–4 the lysates of pCS1-transfected E. coli BL21 cells induced by IPTG at a concentration of 0.5, 0.75, 1, 0 mM, respectively; lane 5 the supernatant of lysate of HBVCS1 expressing E. coli BL21 cells.

was amplified as shown in (Fig. 1A). Insertion of CS1 in the correct direction with the correct open reading frame in the expression vector, pCS1, was confirmed by restriction enzyme digestion identification (Fig. 1A). Direct DNA sequencing of pCS1 was also performed (data not shown).

Six groups of mice were immunized with HBVCS1 emulsified in CFA or IFA, PBS was substitute for HBVCS1 in control groups. Anti-HBc and anti-preS1 could be detected 3 weeks after the first immunization (Fig. 2A). A strong anti-HBc response and moderate anti-preS1 response were observed in mice after immunization with HBVCS1, the highest end point dilution titers of anti-HBc and anti-preS1 were 1:12,800 and 1:3200, respectively (Fig. 2A and B). The titers of antibody were not significantly different between the groups immunized with HBVCS1 plus CFA and those of HBVCS1 plus IFA. However, the titers of anti-HBc were significantly correlated with the immunized HBVCS1 dose and the time after immunization. The major isotype of anti-HBc was IgG2a (IgG2a/IgG1 > 1.5) (data not shown), suggesting that Th1 type of immune responses was induced. The titers of anti-preS1 were also significantly correlated with the time after immunization. There was no significant difference in anti-preS1 between groups immunized with 2 ␮g HBVCS1 and 4 ␮g HBVCS1. 3.4. T-cell proliferation assays of splenocyte of Balb/c mice The splenocytes of mice immunized with HBVCS1 showed significantly higher [3 H] TdR incorporation than those mock immunized when cells were co-cultured in the presence of HBVCS1 (Fig. 2C). While there are differences between the groups of mice immunized with different dose of HBVCS1, there are no significant differences

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between groups of mice immunized with different adjuvant (CFA or IFA). 3.5. Humoral and cellular immune response induced in HBV-Tg mice by HBVCS1 vaccination Anti-HBc and anti-preS1 antibody could be detected on day 21 after first immunization HBVCS1 (group 2). The titers of anti-HBc and preS1 peaked on day 63 with average titer of 2230.84 ± 14.45 and 305.34 ± 3.22, respectively. These titers were sustained to day 84 (2080.65 ± 15.32 and 289.6 ± 3.54) with only slight decrease (Fig. 3A). As expected, the HBV-Tg mice with mock immunization (group 1) have no detectable anti-HBc and anti-preS1 antibody at days 0, 21, 42, 63 and 84. In T-cell proliferation assays, HBV-Tg mice with HBVCS1 immunization elicited significantly higher [3 H] TdR incorporation than those mock immunized when cells were co-cultured in the presence of HBVCS1 (Fig. 3B). In total, HBVCS1 could induce humoral and cellular immune responses in HBV-Tg mice with only slightly less intensity compared to normal Balb/c mice. 3.6. Immunization of HBV-Tg mice with HBVCS1 induced the reduction of HBsAg HBV-Tg mice have high HBsAg titer in their serum before immunization. The titers of HBsAg at day 63 were decreased significantly after two immunizations with HBVCS1 combined with CFA/IFA (Fig. 3C). This was induced specifically by HBVCS1 and not by CFA/IFA alone, since no decrease of HBsAg titer was found in serum of mice in the control group. In addition, this effect lasted a longer time since the titer of HBsAg on day 84 (2.51 ± 0.57) was similar to that of day 63 (2.45 ± 0.7).

Fig. 2. HBVCS1 induced humoral and cellular immune response in Balb/c mice. Groups (eight mice each group) of Balb/c mice were immunized with different doses of HBVCS1 in CFA (group 1, 1 ␮g; group 2, 2 ␮g; group 3, 4 ␮g; group 4, 0 ␮g) or the same doses in IFA (groups 5–8) at day 0, and at day 21 mice were re-immunized with the same dose of HBVCS1, all in IFA. Sera were collected on day 0 (day of first vaccination), and subsequently on days 21, 42 and 63 from first vaccination and stored at −20 ◦ C and tested at the same time; and on days 0 and 21, sera were collected before IP administration. The titers of antibody were expressed as geometry mean±standard deviation. (A) anti-HBc responses to different doses of HBVCS1 immunization. Anti-HBc was undetectable in the sera of mock immunized mice (groups 4 and 8). The titers of anti-HBc were correlated with the dose of HBVCS1 immunized (r = 0.352, P < 0.05) and with time after immunization (r = 0.817, P < 0.01). The titers for the CFA (groups 1–3) and the IFA (groups 4–6) is not significantly different (P > 0.05). (B) Anti-preS1 responses to different doses of HBVCS1 immunization. Result similar to anti-HBc response was induced, but the difference between the titers of anti-preS1 for groups 2 and 3 as well as the difference between groups 6 and 7 is not significant. (C) T-cell proliferation assay of splenocytes of immunized Balb/c mice. Mice were sacrificed at day 63 and the splenocytes were collected for T-cell proliferation assay. T-cell proliferations were expressed as [3 H] TdR uptake corrected for background proliferation in the absence of HBVCS1 ( cpm). Background proliferation ranged from 2100 to 7600 cpm.

3.7. Effect of HBVCS1 immunization on HBV DNA level in serum of HBV-Tg mice Quantitative PCR was performed to detect HBV DNA in sera of the HBV-Tg mice. There were nine mice in HBVCS1 vaccinated group (group 2) and seven mice in control group (group 1), whose sera were HBV DNA positive before vaccination. At day 63, the HBV DNA titer in the mice group 2 was significantly lower than that prior to immunization, and was lower than that of the control group at the same time (Fig. 3D). The difference was still evident on day 84.

4. Discussion The particulate HBcAg is extremely immunogenic and can function both as a T-cell-dependent and T-cell-independent antigen. Immunization with HBcAg preferentially primes the Th1-type cellular immune response [8–11,23]. During chronic HBV infection, HBcAg is the only antigen that was known to elicit a prominent immune response [24].

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Fig. 3. HBVCS1 induced the immune response, reduced the titer of HBsAg and HBV DNA in HBV-Tg mice. Groups of HBV-Tg mice were immunized with 5 ␮g HBVCS1 (group 2, 24 mice) or with PBS (group 1, 16 mice) in CFA at day 0, and at day 21 mice were re-immunized with the same dose of HBVCS1 or PBS in IFA. Sera were collected on days 0, 21, 42, 63 and 84 from the first vaccination, and stored until tested together. Similarly, sera collection on days 0 and 21 was performed before IP administration (A) anti-HBc and anti-preS1 responses to HBVCS1 immunization in HBV-Tg mice. Anti-HBc and anti-preS1 could be detected on day 21, peaked on day 63 and sustained to day 84 with only slightly decrease in mice vaccinated with HBVCS1 (group 2). As expected, no anti-HBc and anti-preS1 response was detected in mock-immunized mice (group 1) at days 0, 21, 42, 63 and 84 (data not shown). (B) T-cell proliferation assay of splenocytes of HBVCS1 immunized HBV-Tg mice. Four mice of group 1 and 12 mice from group 2 were sacrificed at day 63 (week 9) and the splenocytes were collected for T-cell proliferation assay and on day 84 (week 12), another 24 mice (12 in each group) were sacrificed for T-cell proliferation assay. T-cell proliferations were expressed as [3 H] TdR uptake corrected for background proliferation in the absence of HBVCS1 ( cpm). Background proliferation ranged from 2300 to 7500 cpm. (C) HBsAg was determined by the serial end-point dilution method and expressed as logs of geometric mean ± standard deviation. The titer of HBsAg in group 2 at days 63 and 84 was significantly lower than the titer at day 0. The titer of HBsAg in control group at day 84 remained at the same level as at day 0. (D) HBV DNA levels were expressed as log of copies/ml. The HBV DNA levels were mean of nine mice in groups 2 and seven mice in group 1; all of them were HBV DNA positive before immunization. Vaccination with HBVCS1 reduced the HBV DNA level significantly at day 63 in group 2 mice from 5.03 log copies/ml to 3.53 log copies/ml (P < 0.05), while control group 1 mice did not have a significant drop at days 63 and 84. This trend was lasted to day 84 with HBV DNA level of 3.43 ± 0.32 log copies.

In addition, HBcAg is an effective carrier for heterologous peptide epitopes including the HBV surface antigen preS1 and can enhance the immunogenicity of what it carries [13,14]. In doing this, the particle structure of HBcAg is prerequisite for its own immunogenicity [13]. The HBcAg contains two domains. The N-terminal 149 aa domain is required for its oligomerization into capsids and its carboxyl terminal 34–36 aa arginine-rich region non-specifically binds nucleic acids [25]. The truncated HBcAg sequence 1–149 or 1–144 can be efficiently expressed in bacteria and the resulting truncated HBcAg 1–149 or 1–144 arginines can efficiently form particles [26,27]. In this study, we have expressed the fusion protein comprised of HBV core aa 1–155 and preS1 aa 3–55 in E. coli BL21. The fusion gene showed 98.3% homology when aligned with the homologous gene of the HBV adr strain published in Genbank, M38636. The direct sequencing and further immunological assay confirmed that we have correctly cloned and

expressed the fusion protein, HBVCS1. The fusion protein was efficiently expressed in soluble form as indicated by SDS-PAGE analysis. HBVCS1 mostly resident in the supernatant of transformed E. coli BL21 cells lysate (Fig. 1). Further analysis of the antigenicity and immunogenicity of HBVCS1 showed that HBVCS1 bound mAb anti-HBc (Fig. 1C) and mAb anti-preS1 by an ELISA assay. HBVCS1 elicited a strong anti-HBc response and a moderate anti-preS1 response in Balb/c mice (Fig. 2). These results indicated that fusion of preS1 sequence 3–55 to the carboxyl terminus of the truncated HBcAg allowed the fusion protein to maintain its immunogenicity for both HBc and preS1 epitopes. A previous report of Schodel et al. showed that fusion of preS2 133–143 to the carboxyl terminus of HBcAg sequence of 1–156 did not affect its assembly to particles and thus preserved the major HBcAg antibody-binding site and T-cell epitopes [13]. However, our results have gone further, and showed that the fused preS1 epitopes were not

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only surface accessible as assayed by the ELISA assay, but that the fusion protein also preserved its immunogenicity, as it can elicit a moderate anti-preS1 response and a strong anti-HBC in Balb/c mice. This difference from that of others [13,17] may be resulted from the linker GGSG between the carboxyl terminus of HBcAg sequence 1–155 and the preS1 sequence, or else from the different length of preS1 being fused [13,28]. Since HBVCS1 has the immunogenic and related characteristic of HBcAg and preS1, we hypothesized that HBVCS1 may be useful as a therapeutic vaccine of chronic HBV infection. To test this, HBVCS1 with adjuvants CFA/IFA was used to immunize of Balb/c mice and HBV-Tg mice. Strong specific humoral immune responses were induced in Balb/c mice (Fig. 2), and both anti-HBc and anti-preS1 could be detected in sera of mice as early as 3 weeks after their first immunization. Analysis of the isotypes profile of anti-HBc indicated that mice responded mainly with specific IgG 2a antibody production to HBVCS1 vaccination (IgG2a/IgG1 > 1.5). This suggests that the Th1 type of immune responses was induced by vaccination, which is critical for clearance of HBV infection [29]. Although we did not investigate the specific cytokine IFN-␥ release to confirm this, we did find significantly higher HBVCS1-specific T-cell proliferation in mice immunized with HBVCS1 than in those mock immunized (Fig. 2C). More importantly, HBVCS1 vaccination has an effect on reduction of the titer of HBsAg and HBV DNA in sera of HBV-Tg mice (Fig. 3). The immune response is specifically primed by HBVCS1, not by CFA/IFA, since the control groups did not have such responses. The HBV-Tg mice are animal model which mimic HBV asymptomatic human carriers infected at birth, because HBV DNA was integrated into the chromosomes of the mice cell. Our results indicated that HBVCS1 may be the potential therapeutic vaccine for HBV chronic infection. Although immunization with HBVCS1 with adjuvant CFA/IFA did not clear HBsAg and HBVDNA from the sera of HBV-Tg mice, it may be more effective if paired with other more synergistic adjuvant, such as CpG or autologous dendritic cells [12,30]. Recently, Reidl et al. [31] reported that recombinant HBcAg containing only aa 1–144 or aa 1–149 induced an immune response which was Th2 biased compared to with wild type HBcAg, which induced a Th1-biased immune response. They noted that truncated HBcAg with aa 1–144 or aa 1–149 has drastically reduced nucleic acid binding activity (>98%). They hypothesized that the Th1-biased response obtained with wild type HBcAg was facilitated by the trace amount of host bacterial RNA bound to its arginine-rich carboxyl domain and is critical for HBV infection clearance. However, our results indicated that HBVCS1, which contains HBcAg aa 1–155, induced largely Th1-biased response based on analysis of isotypes of anti-HBc in Balb/c mice. In addition, HBVCS1 has an effect on reduction of the titer of HBsAg and HBV DNA in HBV-Tg mice. Thus, HBVCS1 may induce the immune response for HBV

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infection clearance, although the mechanism is not fully understood. The effectiveness of HBVCS1, compared to the recombinant truncated HBcAg containing aa 1–149 of Reidl et al. [31], may due to the fact that HBVCS1 contained more of the arginine-rich domain, which is critical for RNA binding to facilitate priming Th1 immunity. Another reason may be that HBVCS1 contains the fused preS1 aa 3–55, which is immunogenic at both the T and B cell level. The preS1 aa 3–55 domain is crucial for HBV binding to human hepatocyte as well as PBMC. Thus, the preS1 and HBcAg domains of HBVCS1 may have been synergistic in induction of Th1-type immunity against HBV infection. In conclusion, we have expressed the fusion protein HBVCS1 efficiently in E. coli and provided evidence that it has an effect on inhibition of expression of HBsAg and replication of HBV when it is used as therapeutic vaccine. Further study will be needed to address the mechanism of this effect and improve its immunological and virological efficacy against HBV chronic infection. Acknowledgements The authors thank Dr. Shengli Bi and Dr. Yongzhen Jiang (Institute of Virology, Chinese Academy of Preventive Medicine) for their excellent technical assistance; we thank Dr. Chunyan Zhang (Department of immunology, Sun-Yat-Sen University of Medicine) for the gift of anti-IgG1 and anti-IgG2a; we thank Professor Boyu Guo (Second Military Medical University) for providing preS1 antigen. We also thank Dr. Carol Bernstein (Department of Microbiology and Immunology, University of Arizona) for her helpful comment. This work was supported by the municipal government of Shenzhen, China. References [1] Jung MC, Gruner N, Zachoval R, et al. Immunological monitoring during therapeutic vaccination as a prerequisite for the design of new effective therapies: induction of a vaccine-specific CD4+ T-cell proliferative response in chronic hepatitis B carriers. Vaccine 2002;20:3598–612. [2] Yuen MF, Lai CL. Treatment of chronic hepatitis B. Lancet Infect Dis 2001;1:232–41. [3] Pol S, Driss F, Michel ML, Nalpas B, Berthelot P, Brechot C. Specific vaccine therapy in chronic hepatitis infection. Lancet 1994;344:342 [letter]. [4] Pol S, Michel ML, Brechot C. Immune therapy of hepatitis B virus chronic infection. Hepatology 2000;31:548–9. [5] Couillin I, Pol S, Mancini M, et al. Specific vaccine therapy in chronic hepatitis B: induction of T-cell proliferative responses specific for envelope antigens. J Infect Dis 1999;180:15–26. [6] Townsend K, Sallberg M, O’Dea J, et al. Characterization of CD8+ cytotoxic response after genetic immunization with retrovirus vectors expressing different forms of the hepatitis B core and e antigens. J Virol 1997;71:3365–74. [7] Engler OB, Dai WJ, Sette A, et al. Peptide vaccines against hepatitis B virus: from animal model to human studies. Mol Immunol 2001;38:457–65.

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[8] Heathcote J, Mchutchison J, Lee S, et al. A pilot study of C-1899 T-cell vaccine in subjects chronically infected with hepatitis B virus. The CY1899 T-cell vaccine study group. Hepatology 1999;30:531–6. [9] Bertoletti A, Chisari FV, Penna A, et al. Definition of a minimal optimal cytotoxic T-cell epitope within the hepatitis B virus nucleocapsid protein. J Virol 1993;67:2376–80. [10] Ferrari C, Penna A, Bertoletti A, et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J Immunol 1990;145:3442–9. [11] Bertoletti A, Southwood S, Chesnut R, et al. Molecular features of the hepatitis b virus nucleocapsid T-cell epitopes 18–27: interaction with HLA and T-cell receptor. Hepatology 1997;26:1027–34. [12] Bocher WO, Dekel B, Schwerin W, et al. Induction of strong hepatitis B virus specific T helper cell and cytotoxic T lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic HBV infection. Eur J Immunol 2001;31:2071–9. [13] Schodel F, Moriarty AM, Peterson DL, et al. The position of heterologous epitope inserted in hepatitis B virus core particles determines their immunogenicity. J Virol 1992;66:106–14. [14] Clarke BE, Brown AL, Grace KG, et al. presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis B virus core particles produced in bacteria. J Gen Virol 1990;71: 1109–17. [15] Jones CD, Page M, Bacon A, Cahill E, Bentley M, Chatfield SN. T-cell and antibody response characterization of a new recombinant preS1, preS2 and SHBs antigen-containing hepatitis B vaccine: demonstration of superior anti-SHBs antibody induction in responder mice. Vaccine 1999;17:2528–37. [16] Milich DR, McLachlan A, Chisari FV, Kent SB, Thorton GB. Immune response to the preS1 region of HBsAg: a preS1 specific T-cell response can bypass nonresponsiveness to the preS2 and S region of HBsAg. J Immunol 1986;137:315–22. [17] Prange R, Werr M, Birkner M, Hilfrich R, Streeck RE. Properties of modified hepatitis B virus surface antigen particles carrying preS epitope. J Gen Virol 1995;76:2131–40. [18] Ferrari C, Penna A, Bertoletti A, et al. The preS1 antigen of HBV is highly immunogenic at T-cell level in man. J Clin Invest 1989;84:1314–9.

[19] Milich DR. T- and B-cell recognition of hepatitis B virus antigen. Immunol Today 1988;9:380–6. [20] Neurath AR, Kent SB, Strick N. Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell 1986;46:429–36. [21] Paran N, Geiger B, Shaul Y. HBV infection of cell culture: evidence for multivalent and cooperative attachment. EMBO J 2001;16:4443– 53. [22] Neurath AR, Seto B, Strick N. Antibody to synthetic peptides from the preS1 region of the hepatitis B virus envelop (env) protein are virus-neutralizing and protective. Vaccine 1989;7:234–6. [23] Milich DR, Schodel F, Hughes JL, Jones JE, Peterson DL. The hepatitis B virus core and e antigens elicit different Th cell subsets: antigen structure can affect Th cell phenotype. J Virol 1997;71:2192– 201. [24] Milich DR. Immune response to hepatitis B virus: infection, animal model, vaccine. Viral Hepat Rev 1997;3:63–103. [25] Hatton T, Zhou S, Standring DN. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. J Virol 1992;66:5232–41. [26] Kratz PA, Bottcher B, Nassal M. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc Natl Acad Sci USA 1999;96:1915–20. [27] Birnbaum F, Nassal M. Hepatitis B virus nucleotide assembly: primary structure requirements in the core protein. J Virol 1990;64:3319–30. [28] Hui J, Li G, Kong Y, Wang Y. Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitope. J Biotechnol 1999;72:49–59. [29] Hezel F. Th1 and Th2 cells in the cure and pathogenesis of infectious diseases. Curr Opin Infect Dis 1995;8:151–5. [30] Malanchere-Bres E, Payette PJ, Mancini M, Tiollais P, Davis HL, Michel ML. CpG oligodexynucleotides with hepatitis B surface antigen (HBsAg) for vaccination in HBsAg-transgenic mice. J Virol 2001;75:6482–91. [31] Reidl P, Stober D, Oehninger C, et al. Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain. J Immunol 2002;168:4951–9.