Correlation of antiviral T-cell responses with suppression of ... - Nature

Gene Therapy (2006) 13, 1110–1117 & 2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00 www.nature.com/gt

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Correlation of antiviral T-cell responses with suppression of viral rebound in chronic hepatitis B carriers: a proof-of-concept study S-H Yang1, C-G Lee1, S-H Park1, S-J Im1, Y-M Kim1, J-M Son1,2, J-S Wang2, S-K Yoon2, M-K Song3, A Ambrozaitis4, N Kharchenko5, Y-D Yun6, C-M Kim7, C-Y Kim8, S-H Lee8, B-M Kim8, W-B Kim8 and Y-C Sung1 1 Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk, Korea; 2Department of Internal Medicine, College of Medicine, Catholic University of Korea, Seoul, Korea; 3Department of Molecular Microbiology, International Vaccine Institute, Seoul, Korea; 4Department of Infectious Diseases and Microbiology, Vilnius University, Vilnius, Lithuania; 5Department of Gastroenterology and Diet Therapy of Kiev Medical Academy, Kiev, Ukraine; 6Division of Molecular Life Science, Ewha Woman’s University, Seoul, Korea; 7Research Institute, National Cancer Center, Gyeonggi, Korea and 8Research Laboratories, Dong-A Pharm. Co., Ltd, Yongin-si, Gyeonggi, Korea

Despite recent advances in the chemotherapy of chronic hepatitis B (CHB), an effective viral suppression after cessation of therapy has not yet been achieved. To investigate whether hepatitis B virus (HBV)-specific T-cell responses are inducible and can contribute to the viral suppression after cessation of the therapy, we conducted a proof-of-concept study with a DNA vaccine comprising of most HBV genes plus genetically engineered interleukin-12 DNA (IL-12N222L) in 12 CHB carriers being treated with lamivudine (LAM). When the ex vivo and/or cultured IFN-g enzyme-linked immunospot (ELISPOT) assay was performed, the detectable HBV-specific IFN-g secreting T-cell

responses were observed at the end of treatment and during a follow-up. These type 1T-cell responses, particularly CD4+ memory T-cell responses could be maintained for at least 40 weeks after the therapy and correlated with virological responses, but not with alanine aminotransferase elevation. Moreover, DNA vaccination under LAM treatment appeared to be well-tolerated and showed 50% of virological response rate in CHB carriers. Thus, a combination therapy of the DNA vaccine with chemotherapy may be one of new immunotherapeutic methods for the cure of CHB. Gene Therapy (2006) 13, 1110–1117. doi:10.1038/ sj.gt.3302751; published online 9 March 2006

Keywords: hepatitis B virus; DNA vaccine; IL-12; T-cell response; viral suppression

Chronic hepatitis B (CHB) carriers with active viral replication are at a high risk of progressive liver diseases such as cirrhosis and hepatocellular carcinoma.1 Since 350 million people are estimated to be chronically infected with the hepatitis B virus (HBV) worldwide, CHB remains an important global health problem.2 It was reported that complete recovery from acute HBV infection requires cellular immune responses, especially multispecific and polyclonal cytotoxic T lymphocyte (CTL) responses.3 Patients with chronic HBV infection who showed remission also develop vigorous CTL and strong type 1T helper (Th1) immune responses that are comparable to those in patients who have a selflimited disease.4 In contrast, the CTL and Th1 responses are undetectable or relatively weak in patients with chronic HBV infection.3,5 Correspondence: Professor Y-C Sung, Division of Molecular and Life Science, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk, 790-784, Korea. E-mail: [email protected] Received 22 September 2005; revised 2 January 2006; accepted 21 January 2006; published online 9 March 2006

Lamivudine (LAM) and adefovir dipivoxil as nucleoside analogues can suppress HBV replication effectively during treatment period,6,7 but their use is limited by the high risk of viral relapse upon discontinuation even after long-term treatment.2 A restoration of HBV-specific CD4+ and CD8+ T-cell responses by LAM monotherapy was previously observed, but these T-cell responses were not only transient during treatment, but were also undetectable or very weak at the end of 1-year treatment.8,9 It was recently reported that the inverse correlation between the number of antigen-specific interferon (IFN)-g producing CD4+ T cells and serum HBV DNA was observed during the treatment of LAM with recombinant interleukin-12 (IL-12) protein, but not detectable after the treatment.10 Therefore, further studies are needed to elucidate the relationship between T-cell responses and the suppression of viral relapse after stopping the therapy. DNA vaccine has the advantage of inducing both humoral and cellular immune responses, especially Th1 and CTL responses. HBV DNA vaccine was shown to induce strong T-cell responses, leading to the suppression of viral replication in HBV transgenic mice.11 In contrast, DNA immunization induced very weak T-cell

T-cell responses for suppression of viral rebound S-H Yang et al

immune responses (Figure 1a). It was recently demonstrated that broad T-cell response induced by a multigene vaccination during chemotherapy was needed to get a better protection against TB reinfection.16 In addition, a natural form of envelope (Env) gene was divided into the S1/S2 and the S genes, since a DNA vaccine encoding S1/S2/S gene was shown to induce relatively weaker HBsAg-specific responses in mice than the S DNA vaccine.17 The first HB-100 DNA vaccine was injected when the level of serum HBV DNA declined to more than 10-fold of baseline level after LAM treatment (Figure 1b). To evaluate the HBV antigen-specific T-cell responses, we performed an ex vivo IFN-g enzyme-linked immunospot (ELISPOT) assay with overlapping peptide pools covering the HBV Env, core, and polymerase (Pol) sequences. As expected, no significant HBV-specific IFN-g ELISPOT activity (less than 100 spots/106 peripheral blood mononuclear cells (PBMCs)) was observed in the subjects tested before treatment (data not shown), which is consistent with previous reports.3,5,13 However, a detectable ex vivo IFN-g ELISPOT activity to at least one HBV antigen was induced in all seven subjects whose PBMCs were available at the last vaccination (T12) (Figure 2). These results are different from a previous clinical report in which only one out of 10 CHB carriers

responses and showed no suppressive effect on serum HBV DNA and hepatitis B virus surface antigen (HBsAg) in chronically HBV-infected chimpanzees.12 Therefore, it is important to investigate if DNA vaccine can induce detectable T-cell responses and give clinical benefit in humans. In a recent clinical trial, HBV-specific memory T-cell responses could be induced by DNA vaccine alone, but these T-cell responses are relatively weak and transient in CHB carriers.13 As T-cell hyporesponsiveness is associated with high viral loads of HBV,14 lowering the viral load by prior standard LAM treatment could be a rational strategy for optimizing HBV-specific immune responses induced by vaccination in CHB carriers. Here, we investigated if antigen-specific T-cell responses could be safely induced by a combined strategy of DNA vaccination and LAM treatment and if these T-cell responses are responsible for suppression of viral rebound after stopping the therapy in humans. The 12 Caucasian HBV carriers were intramuscularly injected 12 times with HB-100 DNA vaccine at 4-week intervals in combination with 100 mg of LAM (EpivirHBV, GlaxoSmithKline) daily for 5274 weeks and then followed up for another 52 weeks (Table 1 and Figure 1). HB-100 DNA vaccine contains five different plasmids encoding most HBV antigens and a human IL-12N222L DNA15 as a genetic adjuvant to induce broad and strong

a

pGX10 S

gDs

S 1

pGX10 S1/S2/X

1111

226 S1/S2

gDs 1

pGX10 core

X 174/1

154

core 1

pGX10 Pol

183 Pol

gDs HA-e 1

pGX10 hIL-12N222L

832 IRES

hp40(N222L)

MCS SV 40 TPL

CM

V

lyA po

I.E

.

hp35

Enhancer

pGX10 ( 3.64 Kb )

40

SV

b LAM + HB-100 (N=12) *

0 T0

4

8 T1

12 T2

16 T3

20 T4

24 T5

28 T6

32 T7

Treatment period (52 weeks)

36 T8

40 T9

44 48 T10 T11

52 T12

56 F1

64 F3

72 F5

84 F8

92 F10

104 (weeks) F13 (Visits)

Follow-up period (52 weeks)

Figure 1 Schematic diagram of the HB-100 vaccine components and the vaccine regimen. (a) HB-100 consists of five different plasmids (8 mg) including pGX10 S (2 mg)+pGX10 S1/S2/X (1.5 mg)+pGX10 core (1.5 mg)+pGX10 Pol (1 mg)+pGX10 hIL-12N222L (2 mg), which were based on the pGX10 vector.16 Each plasmid in HB-100 was produced from bacterial cells grown in kanamycin selective media under GMP conditions (manufactured by Dong-A pharmaceutical Co., Ltd, Korea). Each HBV gene was amplified with specific primers by polymerase chain reaction (PCR) from adr serotype HBV cDNA.36 The nucleotide sequence and expression of each gene were confirmed by automatic sequencing and ELISA or Western blotting, respectively (data not shown). The corresponding amino-acid numbers of each HBV antigen are indicated. (b) *The first HB-100 injection at T1 was performed at 8 weeks after the start of the LAM treatment except for three subjects; V#118 at week 4, and V#105 and V#106 at week 12. Therefore, V#118 was treated for 48 weeks with LAM, while V#105 and V#106 were treated for 56 weeks. A total 8 mg of HB-100 dissolved in 4 ml of endotoxin-free sodium phosphate buffer solution was injected into two sites (2 ml of HB-100 per site) every 4 weeks as indicated by the arrows to the left and right of musculus gluteus maximus and musculus deltoideus, alternatively. The injection dose and interval of HB-100 were determined by our preliminary results from cynomolgus monkeys (CIT, France) as well as from rats and dogs (Progen, Korea) in terms of immunogenicity and toxicity (data not shown). Gene Therapy


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administered with HBV DNA vaccine alone showed detectable IFN-g secreting T-cell responses.13 This discrepancy may be caused by the presence of LAM treatment or different subjects enrolled; naı¨ve volunteers vs

a

Gene Therapy

b

nonresponders to antiviral therapies (LAM and/or IFNa). Alternatively, vaccination with HB-100 encoding HBV gene (adr) may facilitate the induction of crossreactive T-cell responses in CHB carriers infected with different


types of viruses (ayw or adw). It was reported that immunization with the DNA vaccine encoding HBsAg (adw2), but not with HBsAg (ayw) DNA vaccine, could elicit HBsAg-specific T-cell responses and reduce antigenemia in HBsAg (ayw)-transgenic mice.18 After discontinuation of the combined therapy, six (V#103, V#106, V#113, V#116, V#118, and V#121) out of the 12 subjects showed an undetectable level of viral loads at the end of 52-week follow-up (F13) and thus were designated as virological responders (VRs), although some had transient viral breakthrough during the follow-up period (Table 1 and Figure 2a). The other six subjects, designated as nonvirological responders (NVRs), had a high level of HBV DNA at F13 which is, on average, about six times lower than the pretreatment level, although the viral loads of two NVRs (V#112 and V#114) at F13 appeared to be similar or even higher than those observed at T0 (Table 1 and Figure 2b). It is likely that the virological response rate (50%; six out of 12) in this study is higher than the previous results (19%; five out of 27) as a classical control in which serum HBV DNA was monitored by polymerase chain reaction (PCR) for 24B52 weeks after stopping standard LAM treatment.8,19 Although the virological effect (50%) of the combination therapy observed here is statistically significant compared to that of LAM monotherapy (19%) (w2 4.07, Po0.05), further studies need to be performed through a head-to-head comparison in a large cohort. It was previously reported that the administration of the IL-12 protein alone could inhibit HBV replication,20,21 raising the possibility that the IL-12N222L DNA by itself could be largely responsible for the antiviral effect observed in this study. However, it is unlikely that the hIL-12N222L DNA included in the HB-100 DNA vaccine directly contributes a major effect to the virological responses, because there is a significant difference in the in vivo level of the IL-12 between administrations of recombinant IL-12 protein and of the IL-12 DNA. When mice were intramuscularly injected with a dose of 100 mg (4100 of human dose as calculated with body weight) of the hIL-12N222L DNA, a very low level of peak human IL-12 (2573.2 pg/ml) was detected in the muscle samples only, but not detected in the serum during a month (our unpublished data). None of the pretreatment factors such as age, viral load, and alanine aminotransferase (ALT) appeared to correlate with the induction

of T-cell responses or virological responses (P40.05, data not shown). The longevity of the IFN-g secreting T-cell responses was assessed up to 40 weeks after the last vaccination using ex vivo and/or cultured ELISPOT methods. It has been known that ex vivo ELISPOT assay detects shortlived effector and/or effector memory T cells, whereas cultured ELISPOT activity reflects central memory T-cell responses.22–24 Overall, the VRs had a significantly higher number of IFN-g secreting T cells specific for Env, core, and Pol antigens than the NVRs in ex vivo ELISPOT assays at the end and just after the discontinuation of the treatment (T12 and F1) (Figure 3a). These T-cell responses were also observed at F5 in four out of six VRs except for V#113 and V#118, but none in the NVRs (Figures 2 and 3a). At 40 weeks after the last vaccination (F10), ex vivo ELISPOT activity was nearly undetectable in both VRs and NVRs (Figures 2 and 3a). However, the VRs only showed detectable T-cell responses at F10 as well as F5 in cultured ELISPOT assay (Figures 2a and 3b), indicating that the central memory T-cell responses could persist for at least 40 weeks after the last vaccination. The reason why the sustained memory T-cell responses are induced in this study may be explained by the role of the IL-12N222L DNA. It was previously shown that the IL-12N220L DNA, a murine homologue, as a genetic adjuvant induced codelivered antigen-specific long-term Th1 and CTL responses at a greater degree than wild-type IL-12.15 Furthermore, exogenous IL-12 during antigenic stimulation improved the intrinsic survival properties of T cells, possibly leading to induction of long-lasting memory T-cell responses.25,26 It is worth noting that two NVRs, V#112 and V#114, showed detectable ex vivo ELISPOT activities at T12, but little cultured ELISPOT activity during the follow-up period (Figure 2b). In contrast, all the VRs tested had a relatively high cultured ELISPOT activity during the follow-up period (Figure 2a). These results indicate that the cultured ELISPOT is a better predictor of virological response than ex vivo ELISPOT and that the memory T-cell responses play an important role in the suppression of viral rebound after stopping the therapy. These results agree well with the previous reports that central memory cells with a proliferative potential may be of greater protective importance than effector memory cells in mice and humans.27,28

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Figure 2 The levels of viral load, ALT, HBV antigen-specific T-cell responses, HBeAg, and HBsAg seroconversion in the VRs (a) and in the NVRs (b). To standardize the laboratory tests between the two centers, all the frozen samples were delivered to the central laboratory of Pohang University of Science and Technology in Korea. The HBV markers (HBeAg, HBsAg, anti-HBe, and anti-HBs antibodies) and viral loads were assessed with commercial enzyme immunoassay (EIA) kits (Monolisa, Biorad, Marnes la Coguette, France) and with the Amplicor HBV monitor test (detection limit of 1000 copies/ml, Roche Diagnostic System, Branchburg, NJ, USA). The ALT levels were measured at the participating centers by automated techniques. T0* means the average value of the levels which was measured three times every 2-week interval before the LAM treatment. The ELISPOT assay was performed according to the manufacture’s protocol in the human IFN-g ELISPOT Set (BD Biosciences, San Diego, CA, USA). Briefly, frozen 2 105 PBMCs which were isolated from EDTA-treated blood by Ficoll gradient centrifugation were seeded in triplicates into 96-well plates. The cells were stimulated for 24 h with the peptide pools (1 mg/ml of each peptide) of HBV antigen (adr subtype) derived from 20-mer peptides overlapping by 10 amino acids (aa) that were synthesized by Peptron (www.peptron.co.kr). As a positive control, the ability of the T cells to produce IFN-g in response to concanavalin A (ConA) mitogen was examined. The entire list of peptides can be found in Supplementary Table 2 online. To perform the cultured ELISPOT assay, 1 106 cryopreserved PBMCs were stimulated with HBV and control peptide pools for 10 days on a 24-well plate according to the previous report.24 The criteria for positive ELISPOT results were established by two factors: firstly, the median number of spots in wells containing the HBV peptide pool was at least twice higher than that in control wells containing an irrelevant peptide pool of hepatitis C virus core.37 Secondly, at least 100 spots were detected per 106 PBMCs after background subtraction. Seven seronegative volunteers were additionally recruited and shown to have fewer than 50 spots per 106 PBMCs for each HBV peptide pool (data not shown). The number of spots was enumerated using the AID ELISPOT reader system (AID Gmbh, Strassberg, Germany). #A portion of PBMCs was not available for ELISPOT assay because of low cell viability. Gene Therapy

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Table 1 Virological, biochemical and serological characteristics in chronic hepatitis B carriers treated with lamivudine and HB-100 Pretreatment (T0a)

At the end of treatment (T12)

At the end of follow-up (F13)

Subjectsb

Age

Sex

Viral load (copies/ml)

ALT (U/L)

HBeAg/ anti-HBe

Viral load (copies/ml)

ALT (U/L)

Virological responders

V#103 V#106 V#113c V#116 V#118c V#121d

44 20 20 40 29 49 33.7712.5

M F M F M M

108 000 601 000 12 000 24 333 333 13 467 3 966 667 6 4.8 10 79.7 106

154 372 84 80 94 165 1587111

+/ +/ /+ +/ /+ /+

o1000 3200 o1000 o1000 o1000 o1000

18 30 77 39 39 140 57745

/+ /+ /+ /+ /+ /+

V#105 V#107 V#109d V#110d V#112d V#114

46 20 48 50 47 30 40.1712.2

M M M M M M

9 466 667 986 000 2 200 000 14 013 333 103 000 161 667 4.5 10675.8 106

180 100 126 390 466 63 2217167

+/ +/ /+ /+ /+ +/

o1000 o1000 o1000 o1000 720 000 o1000

40 252 28 29 81 40 78787

/+ +/ /+ /+ /+ +/

Mean7s.d. Nonvirological responders

Mean7s.d.

HBeAg/ Viral load (copies/ml) ALT (U/L) anti-HBe o1000 o1000 o1000 o1000 o1000 o1000 312 000 96 100 432 000 140 000 71 900 3 200 000 5 7.1 10 71.2 106

HBeAg/ anti-HBe

21 16 51 66 104 42 50732

/+ /+ /+ /+ /+ /+

209 161 40 38 42 176 111779

+/ +/ /+ /+ /+ +/

Abbreviations: LAM, lamivudine; M, male; F, female. a T0 represents the average value of the levels measured three times in every 2 week interval before LAM treatment. b Thirteen Caucasian CHB carriers were enrolled from Ukraine (nine subjects) and from Lithuania (four subjects), but one subject from Ukraine was dropped out before vaccination because of house-moving. The major serotypes of the subjects were ayw except two subjects, V#105 and V#1 14 who were adw. All the participants were considered to be horizontally infected with HBV by percutaneous, sexual, or unknown route. As there are several serological and biochemical parameters for the evaluation of liver function and severity of hepatitis in CHB, liver histology was not regarded as an absolute requirement for our proof-of-concept study and thus not performed in this study. Written informed consent was obtained from all trial subjects. This pilot study was approved by the Pharmacological Center of the Ministry of Health in Ukraine and the State Medicine Control Agency of the Ministry of Health in Lithuania and has been conducted in accordance with the ethical principles of the 1975 Declaration of Helsinki. Inclusion criteria: (1) presence of serum HBsAg for at least 6 months; (2) absence of IgM anti-HBc and presence of IgG anti-HBc; (3) elevated serum alanine aminotransferase level higher than 1.5 times the upper limit of the normal range for at least 6 months; (4) positive serum HBV DNA higher than 10 000 copies/ml; (5) negative anti-HIV and anti-HCV status; (6) absence of treatment with antiviral drugs, interferon, corticosteroid or other immune-based therapy within the previous 2 years; (7) absence of alcohol and drug abuse; (8) absence of pregnancy and breast-feeding; (9) absence of hepatocellular carcinoma or decompensated liver disease defined by a serum bilirubin level more than 2.5 times the upper limit of normal, a prothrombin time prolonged by more than 3 s, a serum albumin level lower than 3 g per deciliter, a history of ascites, variceal hemorrhage, or hepatic encephalopathy. c These two subjects are regarded as HBeAg-negative CHB carriers with mild HBV replication (o105 copies/ml). d Four subjects are regarded as true HBeAg-negative patients according to the previous criteria: (1) HBsAg positivity with HBeAg negativity for more than 6 months, (2) increased ALT levels, (3) high serum HBV DNA (4105 copies/ml).38


Group


1115

ISCs/106 PBMCs

a

500 400

P< 0.03

Pol-specific core-specific Env-specific

P< 0.001

300

P< 0.001

200

P> 0.05

100

VR (4) NVR (3) T12 (7)

VR (6) NVR (6)

VR (5) NVR (6)

F5 (12)

F10 (11)

F1 (7) Pol-specific core-specific Env-specific

b 1000

ISCs/106 PBMCs

VR (4) NVR (3)

800 600

P< 0.001 P< 0.001

400 200 0

c 2000 1750 ISCs/106 PBMCs

0

total CD4 depleted CD8 depleted

1500 1250 1000 750 500 250 0 Env core

VR (5) NVR (5)

VR (5) NVR (5)

F5 (10)

F10 (10)

V#116

Pol

Env core

Pol

V#118

Figure 3 (a) Ex vivo and (b) cultured IFN-g ELISPOT responses in the VRs and the NVRs. Data were expressed as mean7s.e.m. and statistical analysis was performed by a Mann–Whitney U test. The number within the parenthesis indicates the number of subjects included in the assay. (c) After stimulation, human anti-CD4 and -CD8 dynabeads (Dynal) and a magnet (Dynal MPC-S) were used for T-cell depletions according to the manufacturer’s instructions. The experiment was performed using PBMCs at F5 from two VRs (V#116 and V#118), as a representation.

The depletion analysis on cryopreserved PBMCs showed that the central memory T-cell responses induced by combination therapy appeared to be mainly CD4+ T-cell-dependent, although a sizeable proportion of the T cells were CD8+ T cells (Figure 3c). These results indicate that antiviral CD4+ memory T cells may play a major role in the long-term suppression of HBV replication. However, further studies including adoptive transfer experiment in CHB patients are needed to clarify which cell types are major determinants responsible for viral suppression. Our data agree with the recent report that the major HBsAg-specific T cells induced by DNA vaccination in CHB patients were CD4+ T cells,13 but not with the other previous result that malaria DNA vaccination mainly induced CD8+ T-cell responses in healthy naı¨ve humans.29 The discrepancy may be caused by the different antigenic nature, the presence or absence of the vaccine adjuvant, and the naı¨ve or antigenexperienced T cells. During the treatment, hepatitis B virus e antigen (HBeAg) seroconversion occurred in four (V#103, V#105, V#106, V#116) out of six HBeAg+ subjects, but the HBeAg reappeared at F5 and viral relapse occurred at F3 in V#105 who showed low levels of ex vivo and cultured ELISPOT activities at T12, F5, and F10. Interestingly, when we examined the HBsAg/anti-HBs status during the whole period of the study, HBsAg seroconversion occurred only in V#106 who had the highest level of memory T-cell responses. The HBsAg seroconversion, an indicator of a cure in HBV infection, was previously

reported to be extremely rare by current LAM monotherapy for 1 year (o1%).6,30 As shown in Figure 2, the induction of IFN-g secreting T-cell response observed in each subjects did not coincide with a high level of ALT, indicating no direct association with the liver damage leading to ALT flare. A significant increase of ALT level in NVRs (V#105 and V#114) appeared to occur just after the viral rebound (Figure 2b), which agrees well with the previous report.8 These results further support the noncytolytic clearance mechanism: HBV could be intracellularly inactivated by IFN-g secreting T cells,31 and cleared from virtually all hepatocytes without massive hepatic destruction in selflimited HBV infection.32 It was also reported that liver injury was caused by a large number of HBV nonspecific inflammatory cells recruited in the liver rather than functionally active HBV-specific CTLs.33 As covalently closed circular (ccc) DNA present in the hepatocytes of CHB carriers produce HBV proteins continuously in the absence of detectable level of HBV DNA in serum,34 it is unlikely that the lack of liver damage is simply due to the suppression of viral replication. Taken together, HBVspecific memory T-cell responses could play a major role in inhibiting viral replication intracellularly without liver damage, leading to the suppression of viral rebound after stopping the combined therapy. However, further studies with a large number of subjects are needed to clearly and quantitatively understand the relative effect of antiviral T-cell responses on pathogenesis and viral clearance in CHB carriers. Gene Therapy


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The incidence rate of adverse events during the treatment with LAM and HB-100 (two out of 12) was similar to that of the adverse events by LAM treatment alone which was previously reported (Supplementary Table 1).30 In addition, there were no severe adverse events such as ALT elevation, depression, and decrease in leukocytes. It was previously reported that intravenous injections of the IL-12 protein to cancer patients showed severe toxicities including the elevation of ALT level with the induction of a high level of serum IFN-g (above 10 000 pg/ml of peak level),35 and that subcutaneous injections of the recombinant IL-12 protein to CHB patients showed mild toxicity, and induced a marginal level of serum IFN-g (about 50 pg/ml),21 indicating that IFN-g is associated with adverse effects. It is worth noting that multiple injections with HB-100 including IL-12N222L DNA showed no detectable serum IFN-g during the whole period of the study in all subjects (detection limit; 47.8 pg/ml) (data not shown). Thus, it is likely that the discrepancy in the adverse effects between our DNA vaccine and previous IL-12 protein therapy may be due to the in vivo level of proinflammatory cytokine, IFN-g. When we further investigated the effect of our aggressive HB-100 DNA vaccination on the induction of autoimmunity, antinuclear/antidouble strand (ds) DNA antibodies as well as antibodies against IL-12N222L were not induced in any of the subjects during (T6, T12) and after (F5, F13) treatment (Supplementary Table 1). To our knowledge, this is the first demonstration that IFN-g secreting memory T-cell responses induced by DNA vaccination under LAM chemotherapy showed a correlation with the long-term suppression of viral replication after stopping the therapy in humans. On the basis of our data, we suggest that HBV DNA vaccine combined simultaneously with standard chemotherapy may be one of novel strategies to efficiently suppress viral relapse, eventually leading to the cure of CHB.

Conflict of interest statement We declare that we have no conflict of interest.

Abbreviations ALT, alanine aminotransferase; CHB, chronic hepatitis B; CTL, cytotoxic T lymphocyte; ELISPOT, enzyme-linked immunospot; Env, envelope; HBeAg, hepatitis B virus e antigen; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; HBc, hepatitis B virus core; HCV, hepatitis C virus; IL-12, interleukin-12; IFN, interferon; ISC, IFN-g secreting cell; LAM, lamivudine; PBMC, peripheral blood mononuclear cell; PCR, polymerase chain reaction; Pol, polymerase

Acknowledgements We thank Dr M Tschaika, E-J Lee, S-Y Eum, and Dr N Opanasyuk for their assistance in processing this study and to Drs J-W Youn and H-T Jin for their revision of the report. This work was supported by grants from National Research Laboratory program of National S&T Program of the Ministry of Science and Technology Gene Therapy

(2000-N-NL-01-C-202), the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (0405DB00-0101-0011), the International Cooperation Research Program of the Ministry of Science & Technology (M60401000227-05A0100-22710), POSCO (2000Y013), and Consortium Project (Genexine Co. Ltd, Daewoong Pharm. Co. Ltd, Dong-A Pharm. Co. Ltd, and POSCO).

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