Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;howeve...Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;however,true functional cure is currently the exception rather than the rule.Nucleic acid vaccines are among the emerging immunotherapies that aim to restore weakened immune function in chronically infected hosts.DNA vaccines in particular have shown promising results in vivo by reducing viral replication,breaking immune tolerance in a sustained manner,or even decimating the intranuclear covalently closed circular DNA reservoir,the hallmark of HBV treatment.Although DNA vaccines encoding surface antigens administered by conventional injection elicit HBVspecific T cell responses in humans,initial clinical trials failed to demonstrate additional therapeutic benefit when administered with nucleos(t)ide analogs.In an attempt to improve vaccine immunogenicity,several techniques have been used,including codon/promoter optimization,coadministration of cytokine adjuvants,plasmids engineered to express multiple HBV epitopes,or combinations with other immunomodulators.DNA vaccine delivery by electroporation is among the most efficient strategies to enhance the production of plasmid-derived antigens to stimulate a potent cellular and humoral anti-HBV response.Preliminary results suggest that DNA vaccination via electroporation efficiently invigorates both arms of adaptive immunity and suppresses serum HBV DNA.In contrast,the study of mRNA-based vaccines is limited to a few in vitro experiments in this area.Further studies are needed to clarify the prospects of nucleic acid vaccines for HBV cure.展开更多
AIM To develop a safe and effective DNAvaccine for inducing humoral and cellularimmunological responses against hepatitis Bvirus surface antigen(HBsAg).METHODS BALB/c mice were inoculated withNV-HB/s,a recombinant pla...AIM To develop a safe and effective DNAvaccine for inducing humoral and cellularimmunological responses against hepatitis Bvirus surface antigen(HBsAg).METHODS BALB/c mice were inoculated withNV-HB/s,a recombinant plasmid that had beeninserted S gene of hepatitis B virus genome andcould express HBsAg in eukaryotes.HBsAgexpression was measured by ABC immunohis-tochemical assay,generation of anti-HBs byELISA and cytotoxic T lymphocyte(CTL),byMTT method,existence of vaccine DNA bySouthern blot hybridization and activation ofoncogene C-myc by in situ hybridization,RESULTS With NV-HB/s vaccination byintramuscular injection,anti-HBs was initiallypositive 2 weeks after inoculation while all micetested were HBsAg positive in the muscles.Thetiters and seroconversion rate of anti-HBs weresteadily increasing as time went on and weredose-dependent.All the mice inoculated with100 μg NV-HB/s were anti-HBs positive onemonth after inoculation,the titer was 1:1024 ormore.The humoral immune response was similarinduced by either intramuscular or intradermalinjection.CTL activities were much stronger(45.26%)in NV-HB/s DNA immunized mice as compared with those(only 6%)in plasma-derived HBsAg vaccine immunized mice.Twomonths after inoculation,all muscle sampleswere positive by Southern-blot hybridization forNV.HB/s DNA detection,but decreased to 25%and all were undetectable by in situhybridization after 6 months.No oncogene C-myc activation was found in the muscle ofinoculation site.CONCLUSION NV-HB/s could generatehumoral and cellular immunological responsesagainst HBsAg that had been safely expressed insitu by NV-HB/s vaccination.展开更多
INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant ...INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].展开更多
AIM To assess the efficacy and safety of in vivo electroporation(EP)-mediated dual-plasmid hepatitis B virus(HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine(LAM) in patients with chronic...AIM To assess the efficacy and safety of in vivo electroporation(EP)-mediated dual-plasmid hepatitis B virus(HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine(LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine(vaccine group, n = 109) or LAM + placebo(control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12(start of treatment with vaccine or placebo, SOT), 16, 24, and 36(end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, morepatients had a decrease in HBV DNA > 2 log10 IU/m L in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir(ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeA g seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy.展开更多
瞄准:为了测试交付为改善作为一个 DNA 疫苗的助手编码 IL-15 的一个原生质标志的可行性,有免疫力的回答由肝炎 B 核心基因 DNA 疫苗导致了。方法:我们使用了 RT-PCR 开发 IL-15 表示构造的基于的策略。我们首先证实基因能由于 IL-15...瞄准:为了测试交付为改善作为一个 DNA 疫苗的助手编码 IL-15 的一个原生质标志的可行性,有免疫力的回答由肝炎 B 核心基因 DNA 疫苗导致了。方法:我们使用了 RT-PCR 开发 IL-15 表示构造的基于的策略。我们首先证实基因能由于 IL-15 的差的表示在埃希氏杆菌属关口 i 被表示。然后, IL-15 原生质标志表示产品的简历活动被 CTLL-2 增长试金识别。从每 IL-15 的 DNA 的 100 微克真核细胞的表示原生质标志和怀有编码 HBV 核心基因(缩短的 pHBc144 ) 的 N 终点的 144 氨基酸的 DNA 的 recombinant 原生质标志习惯于共同使免疫 C57 BL/6 老鼠。anti-HBcIgG 的效价被 ELISA 检测,抗原特定的 CD8 (+) T 房间(CD8 (+) IFN-gamma (+) T 房间) 被在不同时间点染色的细胞内部的 cytokine 检测。结果:在由 pIL-15 和老鼠的抗原特定的 CD8 (+) 房间逐渐地增加了的 pHBc144 DNA 疫苗的合作免疫以后,有免疫力的反应的第一座山峰出现了 14 d 以后,然后,抗原特定的 CD8 (+) T 房间的数字逐渐地减少了并且在 3 瞬间在稳定的水平维持了。在增加以后,抗原特定的 CD8 (+) T 房间的数字到达了第二座山峰与以后的 10 d 一第一山峰双,然后,抗原特定的 CD8 (+) T 房间的数字慢慢地减少了。一个基因助手没在体液的有免疫力的回答上有重要效果的 IL-15 由肝炎 B 核心基因 DNA 疫苗导致了,但是增加了抗原特定的 CD8 (+) T 房间由肝炎 B 核心基因 DNA 疫苗导致了的记忆。结论:HBc Ag 1-144 氨基酸构造的 DNA 疫苗导致交付 plasmid 的 IL-15 提高的有效房间免疫,和 cytokine CD8 (+) T 房间的长寿。展开更多
AIM: To investigate the immunogenicity of a novel DNA vaccine,pSW3891/HBc, based on HBV core gene in Balb/c mice.METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid...AIM: To investigate the immunogenicity of a novel DNA vaccine,pSW3891/HBc, based on HBV core gene in Balb/c mice.METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay.RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine.CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.展开更多
AIM: To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein (HSP) as a strategy to enhance DNA vaccine potency. METHODS: A pCMV-HBeAg-HSP DNA vaccine and a cont...AIM: To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein (HSP) as a strategy to enhance DNA vaccine potency. METHODS: A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was performed in BALB/c mice to monitor anti-tumor immune responses. RESULTS: In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg (44% ± 5% vs 30% ± 6% in E: T > 50:1, P < 0.05). ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe-HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC classⅠor class Ⅱ epitopes derived from HBeAg (74% ± 9% vs 31% ± 6%, P < 0.01). ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV-HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV-HBe-HSP when challenged with CT26-HBeAg.CONCLUSION: The results of this study demonstrate a broad enhancement of antigen-specific CD4+ helper,CD8+ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.展开更多
The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene...The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene on the immunity of HCV multiple-epitope DNA construct in vitro and in vivo in mice. An HCV DNA vector (pVAX1-HBs-MFC) was constructed by fusing HBsAg gene to the N-terminal of an HCV multiple-epitope antigen gene. The pVAX1-HBs-MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blotting. BALB/c mice were intramuscularly immunized with the pVAX1-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum. The cross-reactivity of the antibodies was also checked with two synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-γ production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assays, respectively. The expression of pVAX1-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB/c mice injected with pVAX1-HBs-MFC. The highest titer of antibody against HCV (MFC) was 1∶1280, and the ratio of IgG2a/IgG1 was 1.50±0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the two synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79 ±0.07, and the IFN-γ level was 287±6?pg/ml at week 21 after first immunization. The highest titer of the antibody in control BALB/c mice immunized with pVAX1-MFC was 1∶320, and the ratio of IgG2a/IgG1 was 1.33±0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes cells to MFC was 1.52±0.06, and the IFN-γ level was 225±9.3?pg/ml at week 21 post-immunization. The HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be of potential use in the development of HCV vaccines.展开更多
文摘Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;however,true functional cure is currently the exception rather than the rule.Nucleic acid vaccines are among the emerging immunotherapies that aim to restore weakened immune function in chronically infected hosts.DNA vaccines in particular have shown promising results in vivo by reducing viral replication,breaking immune tolerance in a sustained manner,or even decimating the intranuclear covalently closed circular DNA reservoir,the hallmark of HBV treatment.Although DNA vaccines encoding surface antigens administered by conventional injection elicit HBVspecific T cell responses in humans,initial clinical trials failed to demonstrate additional therapeutic benefit when administered with nucleos(t)ide analogs.In an attempt to improve vaccine immunogenicity,several techniques have been used,including codon/promoter optimization,coadministration of cytokine adjuvants,plasmids engineered to express multiple HBV epitopes,or combinations with other immunomodulators.DNA vaccine delivery by electroporation is among the most efficient strategies to enhance the production of plasmid-derived antigens to stimulate a potent cellular and humoral anti-HBV response.Preliminary results suggest that DNA vaccination via electroporation efficiently invigorates both arms of adaptive immunity and suppresses serum HBV DNA.In contrast,the study of mRNA-based vaccines is limited to a few in vitro experiments in this area.Further studies are needed to clarify the prospects of nucleic acid vaccines for HBV cure.
基金the National Natural Science Foundation of China,No.39670670
文摘AIM To develop a safe and effective DNAvaccine for inducing humoral and cellularimmunological responses against hepatitis Bvirus surface antigen(HBsAg).METHODS BALB/c mice were inoculated withNV-HB/s,a recombinant plasmid that had beeninserted S gene of hepatitis B virus genome andcould express HBsAg in eukaryotes.HBsAgexpression was measured by ABC immunohis-tochemical assay,generation of anti-HBs byELISA and cytotoxic T lymphocyte(CTL),byMTT method,existence of vaccine DNA bySouthern blot hybridization and activation ofoncogene C-myc by in situ hybridization,RESULTS With NV-HB/s vaccination byintramuscular injection,anti-HBs was initiallypositive 2 weeks after inoculation while all micetested were HBsAg positive in the muscles.Thetiters and seroconversion rate of anti-HBs weresteadily increasing as time went on and weredose-dependent.All the mice inoculated with100 μg NV-HB/s were anti-HBs positive onemonth after inoculation,the titer was 1:1024 ormore.The humoral immune response was similarinduced by either intramuscular or intradermalinjection.CTL activities were much stronger(45.26%)in NV-HB/s DNA immunized mice as compared with those(only 6%)in plasma-derived HBsAg vaccine immunized mice.Twomonths after inoculation,all muscle sampleswere positive by Southern-blot hybridization forNV.HB/s DNA detection,but decreased to 25%and all were undetectable by in situhybridization after 6 months.No oncogene C-myc activation was found in the muscle ofinoculation site.CONCLUSION NV-HB/s could generatehumoral and cellular immunological responsesagainst HBsAg that had been safely expressed insitu by NV-HB/s vaccination.
基金Project supported by the grant from Science Foundation of Ministry of Health of China, No. 96-1-347.
文摘INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].
基金Supported by Yigan Biological Products Co.,Ltd.of Guangzhou Pharmaceutical Holdings Ltd.(GPC,Guangzhou,China)Guangdong Provincial Sci.&Tech.Project,No.2012A080204009+2 种基金Guangdong Provincial Natural Science Fund,No.2014A030313 770Guangdong Provincial Public Benefit Foundation,No.2015A010107011National Key Program for Management of AIDS and Viral Hepatitis during the China "11~(th) 5-Year Plan" Period,No.2008ZX10002-003
文摘AIM To assess the efficacy and safety of in vivo electroporation(EP)-mediated dual-plasmid hepatitis B virus(HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine(LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine(vaccine group, n = 109) or LAM + placebo(control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12(start of treatment with vaccine or placebo, SOT), 16, 24, and 36(end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, morepatients had a decrease in HBV DNA > 2 log10 IU/m L in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir(ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeA g seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy.
基金the National High Technology Research and Development Program of China (863 Program), No.G1999054105the National Natural Science Foundation of China, No. 30070693
文摘瞄准:为了测试交付为改善作为一个 DNA 疫苗的助手编码 IL-15 的一个原生质标志的可行性,有免疫力的回答由肝炎 B 核心基因 DNA 疫苗导致了。方法:我们使用了 RT-PCR 开发 IL-15 表示构造的基于的策略。我们首先证实基因能由于 IL-15 的差的表示在埃希氏杆菌属关口 i 被表示。然后, IL-15 原生质标志表示产品的简历活动被 CTLL-2 增长试金识别。从每 IL-15 的 DNA 的 100 微克真核细胞的表示原生质标志和怀有编码 HBV 核心基因(缩短的 pHBc144 ) 的 N 终点的 144 氨基酸的 DNA 的 recombinant 原生质标志习惯于共同使免疫 C57 BL/6 老鼠。anti-HBcIgG 的效价被 ELISA 检测,抗原特定的 CD8 (+) T 房间(CD8 (+) IFN-gamma (+) T 房间) 被在不同时间点染色的细胞内部的 cytokine 检测。结果:在由 pIL-15 和老鼠的抗原特定的 CD8 (+) 房间逐渐地增加了的 pHBc144 DNA 疫苗的合作免疫以后,有免疫力的反应的第一座山峰出现了 14 d 以后,然后,抗原特定的 CD8 (+) T 房间的数字逐渐地减少了并且在 3 瞬间在稳定的水平维持了。在增加以后,抗原特定的 CD8 (+) T 房间的数字到达了第二座山峰与以后的 10 d 一第一山峰双,然后,抗原特定的 CD8 (+) T 房间的数字慢慢地减少了。一个基因助手没在体液的有免疫力的回答上有重要效果的 IL-15 由肝炎 B 核心基因 DNA 疫苗导致了,但是增加了抗原特定的 CD8 (+) T 房间由肝炎 B 核心基因 DNA 疫苗导致了的记忆。结论:HBc Ag 1-144 氨基酸构造的 DNA 疫苗导致交付 plasmid 的 IL-15 提高的有效房间免疫,和 cytokine CD8 (+) T 房间的长寿。
基金Supported by the 135 Project of Jiangsu Province, No. 044
文摘AIM: To investigate the immunogenicity of a novel DNA vaccine,pSW3891/HBc, based on HBV core gene in Balb/c mice.METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay.RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine.CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.
文摘AIM: To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein (HSP) as a strategy to enhance DNA vaccine potency. METHODS: A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was performed in BALB/c mice to monitor anti-tumor immune responses. RESULTS: In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg (44% ± 5% vs 30% ± 6% in E: T > 50:1, P < 0.05). ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe-HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC classⅠor class Ⅱ epitopes derived from HBeAg (74% ± 9% vs 31% ± 6%, P < 0.01). ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV-HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV-HBe-HSP when challenged with CT26-HBeAg.CONCLUSION: The results of this study demonstrate a broad enhancement of antigen-specific CD4+ helper,CD8+ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.
文摘The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene on the immunity of HCV multiple-epitope DNA construct in vitro and in vivo in mice. An HCV DNA vector (pVAX1-HBs-MFC) was constructed by fusing HBsAg gene to the N-terminal of an HCV multiple-epitope antigen gene. The pVAX1-HBs-MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blotting. BALB/c mice were intramuscularly immunized with the pVAX1-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum. The cross-reactivity of the antibodies was also checked with two synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-γ production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assays, respectively. The expression of pVAX1-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB/c mice injected with pVAX1-HBs-MFC. The highest titer of antibody against HCV (MFC) was 1∶1280, and the ratio of IgG2a/IgG1 was 1.50±0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the two synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79 ±0.07, and the IFN-γ level was 287±6?pg/ml at week 21 after first immunization. The highest titer of the antibody in control BALB/c mice immunized with pVAX1-MFC was 1∶320, and the ratio of IgG2a/IgG1 was 1.33±0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes cells to MFC was 1.52±0.06, and the IFN-γ level was 225±9.3?pg/ml at week 21 post-immunization. The HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be of potential use in the development of HCV vaccines.