Liver International ISSN 1478-3223

VIRAL HEPATITIS

Safety and immunogenicity of therapeutic DNA vaccine with antiviral drug in chronic HBV patients and its immunogenicity in mice Seung Kew Yoon1,*, Yong Bok Seo2,*, Se Jin Im2, Si Hyun Bae1, Myeong Jun Song1, Chan Ran You1, Jung Won Jang1, Se Hwan Yang3, You Suk Suh3, Ji Soo Song4, Byong Moon Kim4, Chae Young Kim4, Sook Hyang Jeong5 and Young Chul Sung2,3 1 Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 Division of Molecular and Life Science, Integrative Bioscience & Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Korea 3 Research Institute, Genexine Co. Ltd., Seongnam, Korea 4 Dong-A Pharmaceutical Co. Ltd., Yongin, Korea 5 Department of Internal Medicine, Seoul National University, Bundang Hospital, Seongnam, Korea

Keywords chronic infection – DNA vaccine – hepatitis B – phase I – T-cell response

Correspondence Dr. Young Chul Sung, Division of Molecular and Life Sciences, Integrative Bioscience & Biotechnology, Pohang University of Science and Technology (POSTECH), San-31, Hyojadong, Nam-ku, Pohang 790-784, Korea Tel: +82 54 279 2294 Fax: +82 54 279 5544 e-mail: [email protected] Received 25 October 2013 Accepted 9 March 2014 DOI:10.1111/liv.12530

Abstract Background & Aims: Here, we evaluated the safety and immunogenicity of hepatitis B virus (HBV) DNA vaccine, HB-110, in mice and Korean patients with chronic hepatitis B (CHB) undergoing adefovir dipivoxil (ADV) treatment. Methods: For animal study, mice (BALB/c or HBV transgenic) were immunized with mHB-110, and T-cell and antibody responses were evaluated. For clinical study, 27 patients randomly received either ADV alone or ADV in combination with HB-110. Liver function tests, serum HBV DNA levels and the presence of HBeAg/anti-HBe were analysed. T-cell responses were estimated by ELISPOT and FACS analysis. Results: mHB-110 induced higher T-cell and antibody responses than mHB-100 in mice. No adverse effects were observed by HB-110 cotreated with ADV. HBV-specific T-cell responses were induced in a portion of patients in medium to high dose of HB-110. Interestingly, HB-110 exhibited positive effects on ALT normalization and maintenance of HBeAg seroconversion. One patient, who received high dose of HB-110 exhibited HBeAg seroconversion during vaccination, which correlated with vaccine-induced T-cell responses without ALT elevation. Conclusions: HB-110 was safe and tolerable in CHB patients. In contrast to results in animal models, HB-110 in Korean patients exhibited weaker capability of inducing HBV-specific T-cell responses and HBeAg seroconversion than HB-100 in Caucasian patients. As Asian patients, who are generally infected via vertical transmission, appeared to have higher level of immune tolerance than Caucasian, novel approaches for breaking immune tolerance rather than enhancing immunogenicity may be more urgently demanded to develop effective therapeutic HBV DNA vaccines.

Hepatitis B virus (HBV) is a non-cytopathic virus that induces liver damage via immunopathogenesis. Despite the initial introduction of prophylactic vaccines against HBV in the early 1980s, there remains to be more than 350 million chronic HBV carriers worldwide (1). As the majority of chronic carriers will eventually develop cirrhosis or hepatocellular carcinoma, the ultimate goal of chronic hepatitis B (CHB) treatment is the eradication of HBV or persistent suppression of viral replication. Clinically approved antiviral therapies against chronic HBV infection include pegylated interferon alpha 2a, and nucleos(t)ide analogues including adefovir dipivoxil *These authors contributed equally to this work.

Liver International (2014) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

(ADV), lamivudine (LAM), entecavir, telbivudine and tenofovir (2). However, the major drawbacks of those therapies include serious adverse effects of interferonbased therapies, and emergence of drug resistance during long-term treatment of oral nucleos(t)ide and relapse after its discontinuation because of persistence of HBV covalently closed circular DNA in the nuclei of hepatocytes (3, 4). Therefore, a new therapeutic approach that can safely and completely control HBV replication is urgently required. Recent efforts have been made to develop therapeutic vaccines using HBV protein or DNA to eliminate or continuously suppress HBV infection by enhancing the virus-specific immune response (5, 6).

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Safety and immunogenicity of therapeutic DNA vaccine

Although the precise role of the immune responses in controlling HBV infection is not completely understood, type 1 T-cell (Th1) responses have been known to play a key role in viral clearance via non-cytolytic antiviral mechanism (7, 8). Individuals who demonstrate spontaneous recovery from acute hepatitis B mount a vigorous, multispecific T-cell response against HBV antigens, whereas CHB carriers mount defective cytotoxic T lymphocyte (CTL) (9). Furthermore, hyporesponsiveness of virus-specific CD8+ T cells in CHB is associated with high levels of viral replication (10). Thus, an antiviral strategy for enhancing Th1 and CD8+ CTL responses could be considered for the treatment of patients with chronic HBV infection. Among several vaccination approaches, DNA vaccine has several advantages, including stability and lack of susceptibility to vector specific immunity leading to broad immune responses and repeated immunization. Twenty ongoing clinical studies of HBV DNA therapeutic vaccines are enlisted in the clinical trials.gov database. It was reported that administration of HBV DNA vaccine induced relatively weak and transient HBV-specific memory T-cell responses in CHB carriers (5). The major cause of this hyporesponsiveness might be the immune tolerance because of long-term persistence of large amounts of HBV antigens in the trial subjects (5). Therefore, decrease in viral load by antiviral nucleos(t) ide treatment prior to DNA vaccination may be a rational strategy to achieve more effective immune responses using a HBV DNA vaccine. In the previous report, we generated a therapeutic HBV DNA vaccine named HB-100 and conducted phase I clinical trial in 12 Caucasian patients with CHB (7). Interestingly, HB-100 vaccination was safe and highly immunogenic in CHB Caucasian carriers. To further enhance immunogenicity and efficacy of HB-100 vaccine, we generated HB-110 vaccine by codon optimization and deletion of HBx oncogene from HB-100 vaccine. In vivo pharmacokinetic preclinical studies showed that HB-110 vaccine was rapidly degraded and showed no adverse events (AEs), such as insertional mutagenesis or germline transmission (11). On the basis of these preclinical data, we conducted a phase I clinical trial by escalating doses of HB-110 vaccine plus fixeddose ADV in Korean patients with CHB. Patients and methods Study design and subjects

The randomized, open-label, single-centre, dose-escalation study was designed to evaluate the safety of the HB-110 DNA vaccine combined with the oral nucleotide analogue, ADV. Patients eligible for this study were 19–55 years of age who had been HBsAg positive for ≥6 months, were positive for HBeAg, had received no antiviral therapy for ≥6 months before screening, had >105 HBV DNA copies per millilitre

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of serum and had elevated serum ALT levels between 1.5 and five times the upper limit of normal. Exclusion criteria included those with decompensated liver disease, co-infection with hepatitis C virus (HCV), hepatitis delta virus (HDV) or human immunodeficiency virus (HIV), malignant disease, or an immunosuppressive disease or condition and pregnant or fertile females. During the 14-week screening period, participating patients received ADV daily for 8 weeks; those who showed less than 1/10 the initial viral load were enrolled in this study between July 2007 and April 2010. Of these, 18 patients were administered HB-110 intramuscularly 12 times at 2-week intervals in combination with 10 mg of ADV daily for 24 weeks and then received ADV alone for an additional 24 weeks to minimize the risk of hepatitis flares. Nine patients received only ADV (10 mg) once daily for 48 weeks. Subsequently, 18 patients assigned to the HB-110 plus ADV group were randomly allocated to receive escalating HB-110 doses in combination with fixed doses of ADV (Figure S2). The screening visits (VP1-2) were conducted within 14 weeks of the first HB-110 dose. This study was approved by the ethics committee of the institutional review board of Seoul St. Mary’s Hospital, The Catholic University of Korea and was approved by the Korea Food and Drug Administration. Also, this study was registered on http://www.clinicaltrials.gov (identifier: NCT00513968). All participating patients provided written, informed consent. AEs were defined as any symptom, sign or disease not necessarily related to the treatment or drug, according to the WHO adverse reaction terminology (http://www. umc-products.com/graphics/3149.pdf). The serious adverse event (SAE) was defined as toxicity of WHO grade III or higher. Animals

BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and HBV transgenic mice (12) were kindly provided by Dr. Yamamura K. Mice were housed under specificpathogen-free conditions in an approved animal facility at POSTECH Biotech Center. Construction of HB-110 vaccine

HB-110 vaccine consists as previously described (11). Briefly, pGX10-S/L consists the S and L protein; pGX10C/P consists the core and polymerase protein; pGX10hIL-12 m consists the p35 and p40 subunit of the mutant form of hIL-12 (Figure S1). HB-110 was formulated in 150 mM sodium-phosphate buffer (pH 7.0) at a 2:1:1 ratio of pGX10-S/L:pGX10-C/P:pGX10-hIL-12 m. In preclinical animal model, pGX10-hIL-12 m was replaced with pGX10-mIL-12 m in HB-100 and HB-110 to generate mHB-100 and mHB-110 respectively. Liver International (2014) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Safety and immunogenicity of therapeutic DNA vaccine

Biochemical, serological and virological assessments

Results

Serum ALT, AST, total bilirubin, albumin, prothrombin time and renal function tests (BUN and creatinine) and haematological tests were conducted in a clinical setting. Serological markers (HBsAg, anti-HBs, HBeAg, antiHBe, anti-HCV, anti-HDV and anti-HIV antibodies) were assayed using enzyme immunoassay kits or radioimmunoassay (Abbott Laboratories, Santa Clara, CA, USA). HBV DNA levels were quantified by real-time polymerase chain reaction using Light Cycler (Roche, San Francisco, CA, USA). In addition, HBV genotype was determined using a method described previously (13). Biochemical, haematological and virological assessments were conducted every 4 weeks.

Comparison of immunogenicity between mHB-100 and mHB-110 vaccine in animal model

Measurement of immune response

For mice, splenocytes were stimulated with peptide pools of HBV antigen and interferon (IFN)-c ELISOT assay was performed according to the manufacture’s protocol in the kit (BD Bioscience, San Diego, CA, USA). Antibody responses were determined by an endpoint dilution ELISA assay. ELISA endpoint titres were defined as the highest dilution that yielded an optical density greater than mean plus three times standard deviation of an identically diluted negative control sample. In HBV transgenic mice, positive antibody responses were considered as two-fold higher compared to same age of transgenic mice. For human PBMCs, ELISPOT assay was carried out as previously described (7) with minor modifications. The peptides used for this assay are shown in Table S1. The number of spots was enumerated using the AID ELISPOT reader system (Autoimmun Diagnostika, Strassberg, Germany). IFN-c-secreting cells were expressed as spot-forming cells (SFCs) per 1 9 106 PBMCs. For intracellular cytokine analysis, T cells were expanded in vitro as previously described (14). After expansion, PBMCs were restimulated with or without corresponding peptide pools (envelope, core and polymerase), and cytokine production was evaluated by intracellular staining with anti-IFN-c, anti-CD4, anti-TNF-a, antiCD107a/b (BioLegend, San Diego, CA, USA), anti-CD3, anti-CD8 (BD Biosciences) and LIVE/DEAD (Invitrogen, Grand Island, NY, USA). All samples were acquired with a FACSCanto II flow cytometer (BD Biosciences) and then analysed with FlowJo (Tree Star, Ashland, OR, USA). Polyfunctionality was analysed using SPICE (15).

In the previous study, we demonstrated that HB-100 vaccine was efficiently immunogenic and showed a 50% virological response rate, which correlates with vaccineinduced HBV-specific Th1 responses in CHB carriers. Here, we generated a second generation of HBV DNA vaccine named HB-110 (11) by codon optimization of the HBV envelope proteins (S, L), and core as well as deletion of HBx oncogene to reduce potential risk (Figure S1). It was reported that codon optimization of synthetic genes has been used to elicit stronger immune responses in several species (16). To compare the immunogenicity between mHB-110 and mHB-100 vaccine, immunological tests such as IFN-c ELISPOT and antibody ELISA were performed in normal BALB/c mice which were intramuscularly immunized three times at 2-week intervals. As expected, the T-cell immune responses specific to HBsAg and core were significantly higher in the mice vaccinated with mHB-110 than those vaccinated with mHB-100, indicating improved immunogenicity by codon optimization (Fig. 1A). In addition, the total IgG responses specific to HBsAg and core were markedly increased (Fig. 1B). When we compared mHB-110 with mHB-100 vaccines in the HBV transgenic mice (12) of which HBV whole genome is expressed and thus replicated in a single cycle, both antibody responses against HBsAg of adw subtype employed in DNA vaccine and adr subtype expressed in HBV transgenic mice were higher in the mice immunized with mHB-110 than those with mHB100 (Fig. 1C). In contrast to mHB-100, the HBsAg seroconversion rate was 50% (3/6 mice) in the mice immunized with mHB-110. Taken together, these results provide clear evidence that mHB-110 vaccine is more potent therapeutic vaccine in terms of immunogenicity and efficacy at a greater degree than mHB-100 vaccine in animal models. Patients’ characteristics

Of 31 patients screened, 27 patients with HBeAg-positive chronic HBV infection were randomized. This study included 22 males and five females, with a mean age of 36 years (range 19–55). The baseline demographics, serum ALT, HBV genotype and HBV DNA levels of the patients are summarized in Table 1. Visit compliance and data availability were 96% respectively.

Statistical analysis

The collected data regarding the safety of HB-110 were analysed using descriptive statistical methods. Fisher’s exact test (two sided) was used for comparisons of the groups, and the Mann–Whitney test was used to compare the continuous variables at a significance level of P < 0.05. Liver International (2014) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Safety of the HB-110 vaccine

One (11.1%) of the nine patients in the ADV alone group showed one mild symptom and two SAEs, namely AST and ALT flares, which may have been related to ADV; thus, this patient was dropped out at

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(A)

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(B)

(C)

Fig. 1. Comparison of vaccine-induced cellular and humoral immunity by mHB-100 and mHB-110 in mice. BALB/c mice (n = 6/group) were intramuscularly immunized with 100 lg of mHB-100 or mHB-110 three times with 2-week intervals. Two weeks after the last injection, IFNc responses specific to HBsAg and core were measured (A) and also antibodies specific to HBsAg and core were measured (B). (C) HBV transgenic mice (n = 6/group) were immunized with the same regimen and antibody responses to HBsAg of adw (encoded in DNA vaccine) and adr subtype (expressed in HBV transgenic mice) were determined at week 6. Data are presented as the mean ± SEM and representative of two independent experiments with similar results. S, HBsAg; C, Core. **P < 0.01. Table 1. Baseline characteristics of the patients

Characteristic Age (years) Male sex (%) Height (cm) Body weight (kg) Prior treatment (%) Total bilirubin (mg/dl) Albumin (g/dl) AST (U/L) ALT (U/L) Prothrombin time international normalized ratio Platelet Genotype C (%) HBV DNA (log copies/ml)

ADV (n = 9)

ADV plus HB-110 2 mg (n = 6)

4 mg (n = 6)

8 mg (n = 6)

P value

32.0 (19.0–50.0) 8 (88.9) 173.0 (157.0–178.0) 70.0 (45.0–79.0) 6 (66.7) 0.8 (0.5–1.3) 4.5 (4.2–5.0) 38.0 (21.0–72.0) 58.0 (18.0–141.0) 1.0 (0.9–1.1)

33.5 (30.0–55.0) 5 (83.3) 169.0 (145.0–180.0) 63.0 (54.0–80.0) 3 (50.0) 0.8 (0.6–1.0) 4.3 (3.9–4.7) 28.0 (23.0–49.0) 35.5 (22.0–88.0) 1.0 (1.0–1.1)

38.0 (28.0–47.0) 5 (83.3) 176.0 (162.0–179.0) 75.5 (65.0–86.0) 3 (50.0) 0.7 (0.6–1.3) 4.5 (4.0–4.8) 40.5 (22.0–50.0) 68.5 (11.0–146.0) 1.0 (1.0–1.2)

39.0 (21.0–48.0) 4 (66.7) 169.0 (163.0–180.0) 67.5 (55.0–80.0) 2 (33.3) 1.0 (0.6–1.8) 4.4 (4.4–5.1) 30.5 (23.0–63.0) 52.5 (24.0–108.0) 1.1 (1.0–1.2)

0.7273 0.6361 0.8528 0.7111 0.4197 0.5249 0.4139 0.785 0.8738 0.8311

196.0 (168.0–322.0) 9 (100) 5.5 (2.4–7.2)

248.5 (128.0–262.0) 6 (100) 5.6 (2.5–6.6)

231.5 (204.0–259.0) 6 (100) 5.7 (2.3–6.3)

171.5 (157.0–296.0) 6 (100) 5.4 (2.9–6.6)

0.1731 0.6499

Data present n (%) or median (minimum-maximum). P-values are based on Fisher’s exact test on categorical variables and Mann–Whitney test on continuous variables.

week 20. In contrast, three of the 18 patients (16.6%) experienced mild symptoms: One patient who received 4 mg of HB-110 vaccine plus ADV experienced a mild

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common cold, and two patients (33.3%) who received 8 mg of HB-110 vaccine plus ADV complained of a mild headache, onycholysis, decreased urine flow and Liver International (2014) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Safety and immunogenicity of therapeutic DNA vaccine

Table 2. Frequencies of adverse events (AE) after adefovir dipivoxil (ADV) alone or ADV plus HB-110 vaccination Treatment group

No. of patients

No. of AE

Profile

Grade

Causal relationship

ADV alone (n = 9)

1

3

ADV + HB-110 (4 mg, n = 6) ADV + HB-110 (8 mg, n = 6)

1 2

1 7

ALT elevation AST elevation LDH elevation Flu-like symptoms Headache (94) Onycholysis Decreased urine flow Lymphadenopathy

Severe Severe Mild Mild Mild Mild Mild Mild

Possible Possible Unlikely Unlikely Unlikely Unlikely Unlikely Unlikely

lymphadenopathy; headache and decrease in urine flow were reported to be related to ADV (Table 2). In addition, no skin reaction to local intramuscular injection was observed. No laboratory abnormalities were found in the HB-110 vaccine plus ADV groups. Moreover, no antibody induction against mutant human IL-12 protein occurred in any patients administered the HB-110 vaccine, which agrees well with the previous report using HB-100 vaccine (7). Biochemical, serological and virological responses

We additionally evaluated antiviral and serological responses in the ADV alone and ADV plus HB-110 vaccine groups as a secondary endpoint. Before HB-110 vaccination (0 week), the proportions of patients who achieved ALT normalization (

Safety and immunogenicity of therapeutic DNA vaccine with antiviral drug in chronic HBV patients and its immunogenicity in mice.

Here, we evaluated the safety and immunogenicity of hepatitis B virus (HBV) DNA vaccine, HB-110, in mice and Korean patients with chronic hepatitis B ...
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