Hepatitis B Virus DNA Vaccine Research Articles

Theme: Hepatobiliary system

Theme: Hepatobiliary system

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.

Short noncoding DNA fragments improve the immune potency of electroporation-mediated HBV DNA vaccination.

Electroporation (EP)-mediated DNA immunization can elicit effective immune responses in a variety of animals, and is widely used in research studies and clinical trials. However, high-pulse voltage, high DNA dose and multiple immunizations are still required to achieve considerable immune responses. To further improve the efficiency of EP-mediated DNA immunization, many parameters have been tried and optimized in recent years. In our early research, we found that the short noncoding DNA fragments (sf-DNA) can significantly enhance EP-mediated transgene expression of reporter genes. In this study, we tested the effect of sf-DNA on the immune potency of EP-mediated hepatitis B virus (HBV) DNA vaccination in a mouse model. The results show that the use of sf-DNA in EP-mediated HBV DNA vaccination leads to an enhanced expression of the HBV surface antigen, resulting in higher cellular and humoral responses. Furthermore, the immune responses in the sf-DNA-mediated 120 V cm(-1) EP immunization group were higher than that of the 200 V cm(-1) EP without sf-DNA groups. These data suggest that the sf-DNA can be used as an effective helper molecule to improve the immune response of EP-mediated HBV DNA vaccination, which may make the EP-mediated DNA vaccination more effective and suitable for animal and clinical application.

Cimetidine synergizes with Praziquantel to enhance the immune response of HBV DNA vaccine via activating cytotoxic CD8+ T cell

Previously, we have reported that either CIM or PZQ, 2 clinical drugs, could be used to develop as adjuvants on HBV DNA vaccine to elicit both humoral and cellular immune responses. Here, we demonstrate that combinations of CIM and PZQ as adjuvants for a HBV DNA vaccine, could induce much stronger antigen specific CD4+ and CD8+ T cell responses compared either with CIM or PZQ alone. The synergistic effects of CIM plus PZQ to HBV DNA vaccine were observed on a higher IgG2a/IgG1 ratio, an increase of HBsAg-specific CD4+ T cells capable of producing IFN-γ or IL-17A and a robust IFN-γ-, IL-17A-, or TNF-α-producing CD8+ T cells to HBsAg. Most importantly, the antigen-specific CTL response was also elevated significantly, which is critical for the eradication of hepatitis B virus (HBV) infected cells. Using an HBsAg transgenic mouse model, the expression of HBsAg in the hepatic cells was also significantly reduced after immunized with pCD-S2 in the presence of 0.5% CIM and 0.25% PZQ. Further investigations demonstrated that the synergistic effects of combination of CIM and PZQ were dependent on enhanced cytotoxic CD8+ T cells, which was correlated with impaired activities of regulatory T cells. Therefore, combinations of CIM and PZQ have great potential to be used as effective adjuvants on DNA-based vaccinations for the treatment of chronic hepatitis B.

Mushroom lectin enhanced immunogenicity of HBV DNA vaccine in C57BL/6 and HBsAg-transgenic mice

DNA vaccination is a promising strategy for activating immune responses against hepatitis B virus (HBV) infection. However, the accumulated data have shown that DNA vaccination alone generates weak immune responses. To enhance the immunogenicity of HBV DNA vaccine, lectin purified from pleurotus ostreatus (POL) was used as adjuvant of HBV DNA vaccine for C57BL/6 and HBV surface antigen transgenic (HBVsAg-Tg) mice. Our data demonstrate that low dose of POL (1μg/mouse) in conjunction with HBV DNA vaccine stimulated stronger HBV-specific delayed-type hypersensitivity (DTH) responses and higher HBV-specific IgG level than that in high dose of POL groups (5μg/mouse and 10μg/mouse). POL activated strong Th2 and Tc1 cell responses in immunized C57BL/6 and HBVsAg-Tg mice. POL as adjuvant of HBV DNA vaccine effectively enhanced HBV surface protein antibody (HBVsAb) and decreased HBVsAg level for HBV Tg mice treatment. Furthermore, POL infiltrated more lymphocytes excluding Th1, Th2 and Tc1 cell subtypes to liver of HBVsAg-Tg mice. Together, these results suggest that POL as adjuvant enhanced immunogenicity of HBV DNA vaccination and effectively stimulated immune reaponse for HBsAg-Tg mice treatment. Our findings implicate the potential of mushroom lectin as adjuvant of HBV DNA vaccine.

Astragalus polysaccharides enhance immune responses of HBV DNA vaccination via promoting the dendritic cell maturation and suppressing Treg frequency in mice

Astragalus polysaccharides (APS), an extract from a kind of Chinese traditional herb Astragalus membranaceus, was proved to have strong immunoregulatory properties. In this study, APS was employed as an adjuvant of Hepatitis B virus (HBV) DNA vaccine (pcDS2) and its' effects on immune system of mice were investigated. Our data demonstrated that APS as an adjuvant could increase the HBsAg-specific antibody level as well as the proliferating activity of T cells. APS also could induce CD4+ T cells to produce IL-4, IL-2 and IFN-γ and enhance IFN-γ expression of CD8+ T cells. Moreover, APS could induce the robust activity of the cytotoxic lymphocytes (CTL). Additionally, APS could stimulate the dendritic cells (DC) maturation which is characterized by up-regulation of MHC I/II, CD40, CD80 and CD86, and decreased the frequency of the regulatory T cells (nTreg). Collectively, these findings suggest that APS is a potent adjuvant for the hepatitis B DNA vaccine and can enhance the immune responses of HBV DNA vaccine via promoting DC maturation and inhibit the Treg frequency.

A pilot randomized controlled trial of dual‐plasmid HBV DNA vaccine mediated by in vivo electroporation in chronic hepatitis B patients under lamivudine chemotherapy

A DNA vaccine against the hepatitis B virus (HBV), enhanced by IL-2/IFN-γ fusion protein expression from a plasmid construct and mediated by in vivo electroporation, was evaluated in a total of 39 HBeAg-positive patients with chronic hepatitis B (CHB). The six of 39 patients with a serum alanine aminotransferase (ALT) value of 1-2 times upper limit of normal (ULN) were assigned to the open-label arm (Group01) receiving vaccine monotherapy; the remaining 33 patients with an ALT of more than two times ULN were enroled to the randomized and controlled arm (Group02) receiving lamivudine (LAM) monotherapy (LAM+placebo) or combined therapy (LAM+DNA vaccine) in 1:2 ratio. In Group01, a significant elevation of HBV-specific IFN-γ-secreting T-cell counts in comparison with baseline was observed. In Group02, the proportion of patients with HBV DNA suppression was higher with LAM+DNA vaccine than with LAM monotherapy at each visit time point after the final injection of DNA vaccine at week 36, revealing a significant difference between the two groups (P = 0.03) at week 60. The incidence of dual-site mutations of rtM204/I/S+rtL180M was significantly lower (P = 0.03) with an identified lower virological breakthrough (VBT) rate (P = 0.03) in patients receiving LAM+DNA vaccine than LAM monotherapy, accompanied with a significant higher positive T-cell response rate in patients receiving LAM+DNA vaccine (P = 0.03). In conclusion, this study provides evidence that HBV DNA vaccination is safe and immunologically effective, and that the HBV-specific T-cell responses induced by DNA vaccination under LAM chemotherapy showed a correlation with the suppression of viral replication in patients with CHB.

Praziquantel Facilitates IFN-γ-Producing CD8+ T Cells (Tc1) and IL-17-Producing CD8+ T Cells (Tc17) Responses to DNA Vaccination in Mice

BackgroundCD8+ cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination.Methodology/Principal FindingsHere, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8+ T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8+ T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8+ T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes.Conclusions/SignificanceOur results suggest that PZQ is an effective adjuvant to facilitate Tc1 and Tc17 responses to HBV DNA vaccination, inducing broad CD8+ T cell-based immunotherapy that breaks tolerance to HBsAg.

Ubiquitin Conjugation of Hepatitis B Virus Core Antigen DNA Vaccine Leads to Enhanced Cell-Mediated Immune Response in BALB/c Mice

: Background: Nearly 350 million persons worldwide are chronically infected with hepatitis B virus (HBV). Ubiquitin (Ub) is a highly conserved small regulatory protein, ubiquitous in eukaryotes, that usually serves as a signal for the target protein that is recognised and degraded in proteasomes . The Ub-mediated processing of antigens is rapid and efficient and stimulates cell-mediated immune responses. Accordingly, Ub-mediated processing of antigens has been widely used in chronic-infection and cancer studies to improve immune response.Objectives: Many clinical trials have shown that DNA vaccine potency needs to be greatly enhanced. Here, we report a new strategy for designing an HBV DNA vaccine using the ubiquitin (Ub) sequence. The aim of this study was to investigate a novel DNA vaccination, based on the expression of HBV core antigen (HBcAg), fused to Ub to enhance DNA vaccine potency.Materials and Methods: Mouse ubiquitin fused to the HBcAg gene and cloned into the eukaryotic vector pcDNA3.1 (-). BALB/c mice were immunized with recombinant pUb-HBcAg or pHBcAg DNA vaccine. Lymphocyte proliferation assay, intracellular IFN-γ assay, CTL cytotoxicity assay, and antibody assay were performed to analyze the cellular and humoral immune responses to our DNA constructs.Results: HBcAg was expressed effectively in the COS-7 cells that were transiently transfected with pUb-HBcAg. Strong anti-HBc IgG responses were elicited in mice that were immunized with pUb-HBcAg. The endpoint titers of anti-HBc peaked at 1:656100 on the 42nd day after the third immunization. pUb-HBcAg stimulated greater lymphocyte proliferation and induced higher levels of IL-2 and IFN-γ and a greater percentage of HBcAg-specific CD8+ T cells in mice than pHBcAg. In the CTL assay, the specific lysis rate reached 56.5% at an effector:target ratio of 50:1 in mice that were immunized with pUb-HBcAg.Conclusions: pUb-HBcAg elicits specific anti-HBc responses and induces HBc-specific CTL responses in immunized BALB/c mice. Our results imply that Ub can be used as a molecular adjuvant that enhances the potency of DNA vaccines.Keywords: DNA vaccine; Ubiquitin; Hepatitis B core antigen Implication for health policy/practice/research/medical education:This strategy may have therapeutic value that can be applied to the treatment of infectious diseases. Please cite this paper as:Chen JH, Yu YS, Liu HH, Chen XH, Xi M, Zang GQ, et al. Ubiquitin Conjugation of Hepatitis B Virus Core Antigen DNA Vaccine Leads to Enhanced Cell-Mediated Immune Response in BALB/c Mice. Hepat Mon. 2011;11(8):620-8. [DOI: 10.5812/kowsar.1735143X.689] ©2011 Kowsar M.P.Co. All rights reserved.

Ubiquitin Conjugation of Hepatitis B Virus Core Antigen DNA Vaccine Leads to Enhanced Cell-Mediated Immune Response in BALB/c Mice

Nearly 350 million persons worldwide are chronically infected with hepatitis B virus (HBV). Ubiquitin (Ub) is a highly conserved small regulatory protein, ubiquitous in eukaryotes, that usually serves as a signal for the target protein that is recognised and degraded in proteasomes . The Ub-mediated processing of antigens is rapid and efficient and stimulates cell-mediated immune responses. Accordingly, Ub-mediated processing of antigens has been widely used in chronic-infection and cancer studies to improve immune response. Many clinical trials have shown that DNA vaccine potency needs to be greatly enhanced. Here, we report a new strategy for designing an HBV DNA vaccine using the ubiquitin (Ub) sequence. The aim of this study was to investigate a novel DNA vaccination, based on the expression of HBV core antigen (HBcAg), fused to Ub to enhance DNA vaccine potency. Mouse ubiquitin fused to the HBcAg gene and cloned into the eukaryotic vector pcDNA3.1 (-). BALB/c mice were immunized with recombinant pUb-HBcAg or pHBcAg DNA vaccine. Lymphocyte proliferation assay, intracellular IFN-γ assay, CTL cytotoxicity assay, and antibody assay were performed to analyze the cellular and humoral immune responses to our DNA constructs. HBcAg was expressed effectively in the COS-7 cells that were transiently transfected with pUb-HBcAg. Strong anti-HBc IgG responses were elicited in mice that were immunized with pUb-HBcAg. The endpoint titers of anti-HBc peaked at 1:656100 on the 42nd day after the third immunization. pUb-HBcAg stimulated greater lymphocyte proliferation and induced higher levels of IL-2 and IFN-γ and a greater percentage of HBcAg-specific CD8+ T cells in mice than pHBcAg. In the CTL assay, the specific lysis rate reached 56.5% at an effector:target ratio of 50:1 in mice that were immunized with pUb-HBcAg. pUb-HBcAg elicits specific anti-HBc responses and induces HBc-specific CTL responses in immunized BALB/c mice. Our results imply that Ub can be used as a molecular adjuvant that enhances the potency of DNA vaccines.

Construction of an HBV DNA vaccine by fusion of the GM-CSF gene to the HBV-S gene and examination of its immune effects in normal and HBV-transgenic mice

Background The hepatitis B virus (HBV) DNA vaccine can generate both HBsAg-specific humoral and cellular immune responses. The immune response can be improved by inclusion of an adjuvant, such as the cytokine GM-CSF which is known to be a very good adjuvant. Methods To investigate the ability of GM-CSF to enhance HBV-DNA vaccines, we constructed the plasmids by fusion of GM-CSF gene to the HBV-S gene. Normal and HBV-transgenic mice were then immunized with these plasmids. Results Our results show that pCDNA3.1-GM-CSF-S induced the most powerful HBsAg-specific humoral and cellular immune response, and that it was able to overcome the non-response to HBsAg in HBV-transgenic mice. In contrast, pCDNA3.1-S-GM-CSF was able to induce only a very poor immune response. Conclusions When the HBV-S gene is fused to the GM-CSF gene, the immune effects of the HBV DNA vaccine both in normal and HBV-transgenic mice can be strengthened and HBV-DNA plasmids fused with GM-CSF may be useful for both preventative and therapeutic purposes.

The First Antibody Responses to DNA Vaccines in Humans

To the Editor—In their recent article, Martin et al. [1] report an important phase 1 clinical trial of a DNA vaccine for West Nile Virus. They conclude that “[t]his clinical study represents the first report of neutralizing antibody activity elicited by a DNA vaccine in humans” [1, p. 1739]; the accompanying editorial commentary, by Peterson and Roehrig, repeats this claim [2, p. 1722]. Although Martin et al. elegantly demonstrate neutralizing-antibody responses by using plaque-reduction neutralization and reporter-virus particles, multiple earlier clinical trials demonstrating putative neutralizing-antibody responses have been reported. To our knowledge, the first report of a protective antibody response induced by a DNA vaccine was published in 2000 by Roy et al. [3]. DNA encoding the surface antigen of hepatitis B virus (HBV) was administered to 12 healthy, hepatitis-naive human volunteers via particle-mediated epidermal delivery (PMED). All volunteers developed protective antibody responses, defined as 10 mIU/mL. This level of antibody response against HBV has long been considered sufficient to protect against HBV infection [4]. Subsequent studies confirmed that this HBV DNA vaccine could elicit antibody responses in those who do not respond to conventional vaccine [5] and could increase antibody titers when administered via a disposable, commercial prototype device [6]. These reports, in combination with other clinical studies of DNA vaccines for a variety of pathogens, provide considerable evidence to support further human trials of DNA vaccines. Both the PMED system used in these HBV DNA vaccine studies and the Biojector 2000 NeedleFree Injection Management System that Martin et al. use for West Nile virus are excellent examples of innovative needlefree methods for the delivery of DNA vaccine. These novel delivery methods provide increased hope for the development of a commercially feasible DNA vaccine for use in humans.

Non-cytolytic antigen clearance in DNA-vaccinated mice with electroporation

To explore the potential of electroporation (EP)-mediated hepatitis B virus (HBV) DNA vaccination for the treatment of chronic HBV infection. BALB/c mice were vaccinated with HBV DNA vaccine encoding for the HBV preS(2)-S antigen, combined with or without EP. HBV surface antigen expression plasmid was administered into mice liver via a hydrodynamic injection to mimic HBV infection. The clearance of antigen in the serum and liver was detected by ELISA assay and immunohistochemical staining. The histopathology of the liver tissues was examined by HE staining and serum alanine aminotransferase assay. The immunogenicity of HBV DNA vaccine encoding for the HBV preS(2)- S antigen can be improved by EP-mediated vaccine delivery. The elicited immune responses can indeed reduce the expression of HBV surface antigen (HBsAg) in hepatocytes of the mouse model that was transfected to express HBsAg using the hydrodynamic injection method. The antigen clearance process did not cause significant toxicity to liver tissue, suggesting a non-cytolytic mechanism. The EP-aided DNA vaccination may have potential in mediating viral clearance in chronic hepatitis B patients.

Enhancing the potency of HBV DNA vaccines using fusion genes of HBV‐specific antigens and the N‐terminal fragment of gp96

Many clinical trials show that DNA vaccine potency needs to be greatly enhanced. We have reported that the N-terminal fragment of glycoprotein 96 (gp96) is able to produce an adjuvant effect for production of cytotoxic T-lymphocytes (CTLs) with hepatitis B virus (HBV)-specific peptides. Here, we report a new strategy for HBV DNA vaccine design using a partial gp96 sequence. We linked the N-terminal 1-355aa (N355) of gp96 to HBV genes encoding for structural proteins, the major S and middle S2S envelope proteins and the truncated core HBcAg (1-149aa). ELISPOT, tetramer staining and intracellular IFN-gamma assay were performed to analyze the induced cellular immune responses of our DNA constructs in BALB/c mice and HLA-A2 transgenic mice. The relative humoral immune responses were analyzed in different IgG isotypes. The fusion genes induced 2- to 6-fold higher HBV-specific CD8(+) T cells as compared to the antigens alone. There was an approximate 10-fold decrease in the humoral immune responses with fusion genes based on HBV envelope proteins. Interestingly, the decreased humoral immune responses were not observed when antigens and plasmid encoding N355 were co-delivered. However, an approximate 20-fold higher antibody level was induced when linking N355 to a truncated HBcAg. Immunization by intramuscular injection resulted in predominantly IgG2a antibodies, which indicated that these vaccines preferentially prime Th1 responses. We constructed highly immunogenic fusions by linking the N-terminal fragment of gp96 to HBV antigens. Our results imply that the N-terminal fragment of gp96 may be used as a molecular adjuvant to enhance the potency of DNA vaccines.

A truncated C-terminal fragment of Mycobacterium tuberculosis HSP70 gene enhanced potency of HBV DNA vaccine

DNA vaccine represents an attractive approach to therapy of chronic hepatitis B virus (HBV) infection because of its ability to generate antigen-specific immunity; nevertheless, there is still a need to increase the potency of DNA vaccine. Mycobacterium tuberculosis heat shock protein70 (HSP70) has both chaperon and cytokine functions, and has been shown to act as an adjuvant when co-administered with peptide antigens or given as fusion proteins. Here we evaluated the effects of two truncated HSP70 molecules, N-terminal domain (HSP70 1–360, amino acids 1–360) and C-terminal domain (HSP70 359–610, amino acids 359–610) of mycobacterial HSP70, on the potency of antigen-specific immunity generated by a HBV DNA vaccination. We found that only the HSP70 359–610-fused HBV DNA vaccination resulted in a significant increase in hepatitis B surface antigen (HBsAg)-specific humoral response, while the HSP70 1–360- or the complete HSP70 molecule-fused vaccine did not. Moreover, HSP70 359–610-fused DNA vaccine did not induce anti-HSP70 antibody. Interestingly, HSP70 359–610 not only enhanced HBsAg-specific cytotoxic lymphocytes (CTL) responses but also overcame the epitope suppression caused by L d-restricted epitope. Meanwhile, HSP70 369–610 mediated T helper (Th) cell balance towards Th1 pathway. In a HBV transgenic mouse model, the HSP70 359–610 fusion vaccine facilitated clearance of circulating HBsAg and down-regulation of HBV replication. These results suggested that the truncated mycobacterial HSP70 molecule, HSP70 359–610, might be a superior candidate to deliver the adjuvant function in HBV DNA vaccination instead of the complete HSP70 molecule.

A novel HBV DNA vaccine based on T cell epitopes and its potential therapeutic effect in HBV transgenic mice

DNA vaccination represents a novel therapeutic strategy for chronic hepatitis B virus (HBV) infection. Recently, some HBV DNA vaccines have been used in the preliminary clinical trials and exhibited exciting results in chronic HBV carriers. But these vaccines only encoded the single viral antigen, the S or the PreS2/S antigen. In this study, we designed a polytope DNA vaccine encoding multiple T cell epitopes. We found that it induced stronger CTL responses than the vaccine encoding the single antigen in H-2d and H-2b mice, although the CTL response to Ld-restricted epitope suppressed the CTLs to other epitopes in H-2d-restricted mice. Interestingly, heat shock protein 70 as an adjuvant not only enhanced CTL response to the viral antigen but also overcame this epitope suppression. Furthermore, the polytope DNA vaccine resulted in a long-term down-regulation of hepatitis B virus surface antigen and inhibition of HBV DNA replication in a HBV transgenic mouse model. Therefore, our research indicates that it is practicable and feasible to design a polytope DNA vaccine for chronic hepatitis B immunotherapy.

Enhancement of immune responses to an HBV DNA vaccine by electroporation

These studies document the ability of electroporation (EP)-based DNA vaccination to induce multi-specific CTL responses to hepatitis B virus (HBV) DNA vaccination in normal mice and marked immune responses to multivalent HBV DNA immunization in larger animal species. These results suggest that electroporation-mediated HBV DNA vaccination is worth pursuing as a treatment for chronic HBV infection.

Development of Th1 and Th2 Populations and the Nature of Immune Responses to Hepatitis B Virus DNA Vaccines Can Be Modulated by Codelivery of Various Cytokine Genes

In this study, we provide direct evidence that the magnitude and nature of the immune response to a DNA vaccine can be differentially regulated by codelivery of various mouse cytokine genes. Mice immunized with a hepatitis B virus (HBV) DNA vaccine and the IL-12 or IFN-gamma gene exhibited a significant enhancement of Th1 cells and increased production of anti-HBV surface IgG2a Ab, as well as a marked inhibition of Th2 cells and decreased production of IgG1 Ab. In contrast, coinjection of the IL-4 gene significantly enhanced the development of specific Th2 cells and increased production of IgG1 Ab, whereas Th1 differentiation and IgG2a production were suppressed. Coinjection of the IL-2 or the granulocyte-macrophage-CSF gene enhanced the development of Th1 cells, while the development of Th2 cells was not affected, and the production of IgG1 and IgG2a Ab were both increased. The CTL activity induced by HBV DNA vaccination was most significantly enhanced by codelivery of the IL-12 or IFN-gamma gene, followed by the IL-2 or granulocyte-macrophage-CSF gene, whereas codelivery of the IL-4 gene suppressed the activity. When challenged with HBV surface Ag (HBsAg)-expressing syngeneic tumors, significant reduction of tumor growth was observed in mice that were coadministered the IL-12 gene but not the IL-4 gene. Taken together, these results demonstrate that application of a cytokine gene in a DNA vaccine formulation can influence the differentiation of Th cells as well as the nature of an immune response and may thus provide a strategy to improve its prophylactic and therapeutic efficacy.