Induction of multispecific Th-1 type immune response against HCV in mice by protein immunization using CpG and Montanide ISA 720 as adjuvants

Recent studies demonstrate that Th1-type immune responses against a broad spectrum of hepatitis C virus (HCV) gene products are crucial to the resolution of acute HCV infection. We investigated new vaccine approaches to augment the strength of HCV-specific Th1-type immune responses. ELISPOT assay revealed that single or multiple protein immunization using both CpG ODN and Montanide ISA 720 as adjuvants induced much stronger IFN-gamma-producing Th1 responses against core, NS3 and NS5b targets than did the formulation without these adjuvants. Protein vaccination using CpG ODN and Montanide ISA 720 as adjuvants also greatly enhanced humoral responses to HCV core, E1/E2 and NS3. When specific IgG isotypes were assayed, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants produced higher titers of IgG2a dominant antibodies than did protein immunization alone, indicating a more Th1-biased pathway. This increase in IgG2a is consistent with the induction of Th1 cells secreting IFN-gamma demonstrated by ELISPOT assay. In conclusion, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants greatly enhanced cellular (Th1 type) as well as humoral immune responses against HCV in Balb/c mice. The use of adjuvants appears critical to the induction of Th1 immune responses during HCV vaccination with recombinant proteins.     

Hepatitis C virus soluble E2 in combination with QuilA and CpG ODN induces neutralizing antibodies in mice

Several studies have emphasized the importance of an early, highly neutralizing antibody response in the clearance of Hepatitis C virus (HCV) infection. The envelope glycoprotein E2 is a major target for HCV neutralizing antibodies. Here, we compared antibody responses in mice immunized with native soluble E2 (sE2) from the H77 1a isolate coupled with different adjuvants or combinations of adjuvants. Adjuvanting sE2 with Freund's, monophosphoryl lipid A (MPL), cytosine phosphorothioate guanine oligodeoxynucleotide (CpG ODN), or alpha-galactosylceramide (αGalCer) derivatives elicited only moderate antibody responses. In contrast, immunizations with sE2 and QuilA elicited exceptionally high anti-E2 antibody titers. Sera from these mice effectively neutralized HCV pseudoparticles (HCVpp) 1a entry. Moreover, the combination of QuilA and CpG ODN further enhanced neutralizing antibody titers wherein cross-neutralization of HCVpp 4 was observed. We conclude that the combination of QuilA and CpG ODN is a promising adjuvant combination that should be further explored for the development of an HCV subunit vaccine. Our work also emphasizes that the ideal combination of adjuvant and immunogen has to be determined empirically.     

Cationic microparticles are a potent delivery system for a HCV DNA vaccine

We initially evaluated in mice the ability of naked DNA encoding intracellular forms of the E1E2 envelope proteins from HCV to induce antibody responses and compared the responses induced with the same plasmid adsorbed onto cationic poly (lactide co-glycolide) (PLG) microparticles. Although naked DNA was only able to induce detectable responses at the 100 microg dose level, making this approach impractical for evaluation in larger animals, PLG/DNA induced detectable responses at 10 microg. In addition, the PLG/DNA microparticles induced significantly enhanced responses to naked DNA when compared at the same dose level. Remarkably, PLG/DNA induced comparable responses to recombinant E1E2 protein adjuvanted with the emulsion MF59. Furthermore, PLG/DNA effectively primed for a booster response with protein immunization, while naked DNA did not. Therefore, PLG/DNA was selected for further evaluation in a non-human primate model. In a study in rhesus macaques, PLG/DNA induced seroconversion in 3/3 animals following three immunizations. Although the antibody responses appeared lower than those induced with recombinant protein adjuvanted with MF59, following a fourth dose, PLG/DNA and protein induced comparable responses. However, a single booster dose of recombinant protein administered to the animals previously immunized with PLG/DNA induced much higher responses. In addition, one of three animals immunized with PLG/DNA showed a cytotoxic T lymphocyte response in peripheral blood lymphocytes. In conclusion, cationic PLG microparticles with adsorbed HCV DNA generates potent immune responses.     

Oral immunization with attenuated Salmonella carrying a co-expression plasmid encoding the core and E2 proteins of hepatitis C virus capable of inducing cellular immune responses and neutralizing antibodies in mice

Hepatitis C virus (HCV) core protein has long been considered an attractive candidate for inclusion in a protective vaccine. However, this protein may hamper the development of systemic immune responses because of its immune suppressive properties. We previously reported that immune responses to HCV core protein could be efficiently induced by attenuated Salmonella carrying the HCV core protein, but not the HCV core DNA vaccine. To optimize the combination of the core protein and envelope protein 2 (E2) into a vaccine formulation to induce cellular immune responses and neutralizing antibodies, we constructed a plasmid containing two expression cassettes. One expression cassette was included to regulate the expression of HCV core protein by an inducible in vivo-activated Salmonella promoter, the other was included to regulate the expression of HCV E2 protein by the cytomegalovirus enhancer/promoter. Oral immunization of BALB/c mice with the attenuated Salmonella strain SL7207 carrying this plasmid efficiently induced HCV core and E2-specific cellular immune responses and antibodies. IgG purified from immunized mice could neutralize the infectivity of HCV pseudoparticles (HCVpp) of both the autologous Con 1 isolate and the heterologous H77 isolate, and cell culture produced HCV (HCVcc) of Con1-JFH1 chimera. These results indicated that this vaccine strategy can effectively deliver core and E2 protein to the immune system and provide a promising approach for the development of prophylactic and therapeutic vaccines against HCV infection.     

A single immunization with a plasmid encoding hepatitis C virus (HCV) structural proteins under the elongation factor 1-alpha promoter elicits HCV-specific cytotoxic T-lymphocytes (CTL).

Recent studies have raised the possibility that DNA-based vaccination may prove useful for generating virus-specific cytotoxic T-lymphocytes (CTL) responses. Recently, a plasmid containing the human elongation factor 1alpha(EF1-alpha) promoter, pEF321, was reported to be a versatile expression vector for gene expression in mammalian cells in vitro. In the present study, we assessed the capability of a novel plasmid, pEFCE1E2, encoding hepatitis C virus (HCV) structural proteins (core, E1 and E2) under the EF1-alpha promoter to generate CTL against HCV in vivo. BALB/c mice were immunized with the pEFCE1E2 but not with a plasmid possessing the same cDNA under the cytomegalovirus developed HCV-specific effector cells by a single immunization. These effector cells elicited by pEFCE1E2 immunization were CD8(+) and major histocompatibility complex class I restricted. These studies provided evidence for the potential utility of the EF1-alpha promoter for development of DNA vaccines against HCV infections.     

Immunogenicity of the E1E2 proteins of hepatitis C virus expressed by recombinant adenoviruses

The E1 and E2 proteins of hepatitis C virus (HCV) are believed to be the viral envelope glycoproteins that are major candidate antigens for HCV vaccine development. We reported previously that the replication-competent recombinant adenovirus encoding core-E1-E2 genes of HCV (Ad/HCV) produces serologically reactive E1 and E2 proteins forming a heterodimer in substantial amounts. Here, we examined immunogenicity of the E1E2 proteins copurified from HeLa cells infected with Ad/HCV virus in mice. Furthermore, we constructed a replication-defective recombinant adenovirus encoding the core-E1-E2 genes of HCV (Ad.CMV.HCV) and examined immunogenicity of the virus in mice. The mice immunized intraperitoneally with the copurified E1E2 proteins induced mainly antibodies to E2, but not to E1 by Western blot analysis. The sera of mice immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81. E2-specific cytotoxic T cells (CTLs), but not antibodies to the E1E2 antigens were induced in the mice intramuscularly immunized with Ad.CMV.HCV virus. When immunized with both Ad.CMV.HCV virus and the E1E2, mice elicited E2-specific CTLs and antibodies to the E1E2 antigens. The results suggest that immunization of Ad.CMV.HCV virus combined with E2 protein is an effective modality to induce humoral as well as cellular immune response to E2 antigen.     

Enhancement of the immunity to foot-and-mouth disease virus by DNA priming and protein boosting immunization.

Subunit vaccination is effective in eliciting humoral responses to a variety of viral antigens, however, it has not generated persistent protective immunity to foot-and-mouth disease virus (FMDV). In this study, we observed that priming mice with a DNA plasmid encoding VP1 of the FMDV O/Taiwan/97 capsid protein followed by boosting with a VP1 peptide conjugate (P29-KLH) resulted in production of not only high titers of antibodies but also antibodies with FMDV neutralizing activities. Moreover, the mice immunized in this manner cleared the virus from their sera in FMDV challenge experiments. Mice subjected to DNA plasmid priming and P29-KLH protein boosting had relatively higher ratio of IgG2a/IgG1 than those primed and boosted with P29-KLH conjugate. Addition of an oligodeoxynucleotide (ODN) containing immunostimulatory cytosine-phosphate-guanosine (CpG) motifs to P29-KLH conjugate also induced a higher ratio of IgG2a/IgG1 and significantly higher titer of neutralizing antibodies. These results indicate that treating animals with DNA plasmids priming and FMDV antigen(s) boosting may elicit immunity to FMD and this immune response may be augmented by CpG ODN.     

Efficient induction of T helper 1 CD4+ T-cell responses to hepatitis C virus core and E2 by a DNA prime-adenovirus boost

Hepatitis C virus (HCV) is an important causative agent of liver disease, but currently there is no available prophylactic vaccine against HCV infection. Here, we investigated the HCV E2- and core-specific T-cell responses induced by DNA (D) and/or recombinant adenovirus (A) vaccines. In single (D versus A) or double immunizations (D-D versus A-A), the recombinant adenovirus vaccines induced higher levels of IFN-gamma secreting T-cell response and cytotoxic T lymphocytes (CTL) response than the DNA vaccines. However, a heterologous (D-A) regimen elicited the highest level of T helper 1 (Th1) CD4(+) T-cell responses. Furthermore, three E2-specific CTL epitopes were mapped using a peptide pool spanning the E2 protein sequence (a.a. 384-713) in BALB/c mice, and one of these (E2 405-414: SGPSQKIQLV) was shown to be immunodominant. Interestingly, no significant differences were found in the repertoire of E2-specific T-cell responses or in the immunodominance hierarchy of the three epitopes induced by D-D, D-A, A-A, and A-D, indicating that the breadth and hierarchy of T-cell responses is independent of these different vaccination regimens. In conclusion, the heterologous DNA prime-recombinant adenovirus boost regimen described offers an efficient promising strategy for the development of an effective T-cell-based HCV vaccine.     

Immunity induced by DNA immunization with herpes simplex virus type 2 glycoproteins B and C

The complete sequence of herpes simplex virus type 2 (HSV-2) glycoproteins B and C (gB & gC) were cloned into plasmid expression vectors and evaluated in murine and guinea pig genital HSV-2 models. Balb/c mice were immunized with either pgB-2 or pgC-2 plasmids intramuscularly (IM) or intradermally (ID). The vaccines induced HSV-2-specific neutralizing and ELISA IgG antibody, but little or no enhancement of viral clearance from the vagina was detected following intravaginal challenge. Immunization of guinea pigs with pgB-2 or pgC-2 induced ELISA IgG antibody; however, antibody titers were approximately one log(10) unit lower than that seen in HSV-2 convalescent sera. IM immunization of guinea pigs with either plasmid also did not decrease vaginal viral shedding following vaginal challenge, but the severity of the acute disease and the subsequent number of recurrent lesion days were reduced in animals immunized with pgB-2. Lastly, IM immunization of latently infected guinea pigs with a combined gB-2 and gC-2 plasmid vaccine significantly reduced the number of subsequent HSV-2 recurrences. DNA vectors expressing gB-2 or gC-2 were both immunogenic, although the gB-2 plasmid induced higher titers of antibody and significantly reduced primary and recurrent herpetic disease in the guinea pig model. These results also suggest that immunotherapy with plasmid expression vectors may be effective against recurrent genital HSV-2 disease.     

Immunization with DNA, adenovirus or both in biodegradable alginate microspheres: effect of route of inoculation on immune response

To determine the potential for biodegradable alginate microspheres to be used as a delivery vehicle for DNA based vaccines, we constructed a plasmid, pMNe-gal-SV40, containing the bacterial beta-galactosidase (LacZ) gene under the control of the murine cytomegalovirus (MCMV) immediate-early promoter and the simian virus 40 (SV40) polyadenylation signal. The effect of the route of administration and co-administration of adenovirus on systemic and mucosal immune responses were investigated. Mice were inoculated orally, intranasally (i.n.), intramuscularly (i.m.), subcutaneously (s.c.) or intraperitoneally (i.p.) on days 0, 14 and 28 with microspheres containing plasmid DNA, bovine adenovirus type 3 (BAd3) or plasmid DNA + BAd3. Systemic routes of immunization (i.m., s.c. and i.p.) resulted in higher LacZ- or BAd3-specific IgG ELISA titers compared to those obtained by mucosal routes of inoculation (oral and i.n.). Mucosal immunization led to slightly higher titers of LacZ- or BAd3-specific IgA at mucosal sites compared to those obtained by the various systemic routes. All the routes of immunization induced LacZ-specific lymphoproliferation. Co-administration of BAd3 enhanced the LacZ-specific IgG response irrespective of the route of administration.     

DNA vaccine expressing the non-structural proteins of hepatitis C virus diminishes the expression of HCV proteins in a mouse model

Most of the people infected with hepatitis C virus (HCV) develop chronic hepatitis, which in some cases progresses to cirrhosis and ultimately to hepatocellular carcinoma. Although various immunotherapies against the progressive disease status of HCV infection have been studied, a preventive or therapeutic vaccine against this pathogen is still not available. In this study, we constructed a DNA vaccine expressing an HCV structural protein (CN2), non-structural protein (N25) or the empty plasmid DNA as a control and evaluated their efficacy as a candidate HCV vaccine in C57BL/6 and novel genetically modified HCV infection model (HCV-Tg) mice. Strong cellular immune responses to several HCV structural and non-structural proteins, characterized by cytotoxicity and interferon-gamma (IFN-γ) production, were observed in CN2 or N25 DNA vaccine-immunized C57BL/6 mice but not in empty plasmid DNA-administered mice. The therapeutic effects of these DNA vaccines were also examined in HCV-Tg mice that conditionally express HCV proteins in their liver. Though a reduction in cellular immune responses was observed in HCV-Tg mice, there was a significant decrease in the expression of HCV protein in mice administered the N25 DNA vaccine but not in mice administered the empty plasmid DNA. Moreover, both CD8⁺ and CD4⁺ T cells were required for the decrease of HCV protein in the liver. We found that the N25 DNA vaccine improved pathological changes in the liver compared to the empty plasmid DNA. Thus, these DNA vaccines, especially that expressing the non-structural protein gene, may be an alternative approach for treatment of individuals chronically infected with HCV.     

合并感染HIV对1b型HCV E2/NS1包膜区基因影响

目的 研究人类免疫缺陷病毒(HIV)感染对1b型丙型肝炎病毒(HCV) E2/NS1包膜区基因变异的影响,探讨2组间E2/NS1区基因序列同源性差异,为HCV/HIV合并感染者中HCV的治疗提供依据.方法 对河南省某有偿献血村进行随访,将得到的所有HCV阳性病例255例根据合并HIV感染的情况分成2组,并对其基因分型,然后进行逆转录( RT)-巢式PCR扩增1b型HCV E2/NS1包膜区基因,继而进行单链构象多态性分析(SSCP)、纯化后测序.结果 HCV单纯感染组和HIV/HCV合并感染组SSCP平均条带数分别为(3.4±0.55)、(2.6±0.55)条,差异有统计学意义(t =2.32,P =0.049);HCV单纯感染组和HIV/HCV合并感染组HCV E2/NS1包膜区同源性分别为76.7％和87.6％,差异有统计学意义(x2=20.13,P＜0.001);2组第一高变区(HVR-1)同源性分别为59.3％和81.9％,差异有统计学意义(x2=10.39,P=0.001);2组第3高变区(HVR-3)同源性分别为71.7％和83.9％,差异有统计学意义(x2=4.60,P=0.03);单纯HCV组中变异性较高的氨基酸位点在HCV/HIV合并感染组中表现出较高的保守性.结论 HIV感染对1b型HCV E2/NS1包膜区基因变异具有抑制作用,且其主要体现在HVR-1和HVR-3区.     

A novel adjuvant for vaccine development in the aged

A conformationally-biased, response-selective agonist of human C5a(65-74) (EP67) activated antigen presenting cells (APC) from aged C57Bl/6 mice in vitro and the generation of antigen (Ag)-specific antibody (Ab) responses in aged mice in vivo. EP67, induced the release of the pro-inflammatory cytokines IL-6, TNFα, and INFγ from splenic APCs obtained from both aged and young mice. Both aged and young mice produced high Ag-specific IgG Ab titers when immunized with EP67-containing vaccines to ovalbumin (OVA-EP67) and to a protein (rPrp1) from the cell wall of Coccidioides (rPrp1-EP67). Immunization with EP67-containing vaccines resulted in higher IgG titers in both young and aged mice compared to mice immunized with OVA adsorbed to alum (OVA/alum) and Prp1 admixed with CpG (rPrp1 +CpG). Aged and young mice immunized with the EP67-containing vaccines generated higher titers of IgG1 and IgG2b relative to their aged-matched counterparts immunized with OVA/alum or Prp1 +CpG. These results indicate that EP67 induces humoral immunity in aged mice not obtainable with alum and CpG. These results support the use of EP67 as a potential vaccine adjuvant suited to the elderly.     

Recombinant retrovirus-derived virus-like particle-based vaccines induce hepatitis C virus-specific cellular and neutralizing immune responses in mice

While the immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood, it is now admitted that an effective vaccine against HCV will need to induce both cellular and humoral immune responses and address viral heterogeneity to prevent immune escape. We developed a vaccine platform specifically aimed at inducing such responses against HCV antigens displayed by recombinant retrovirus-based virus-like particles (VLPs) made of Gag of murine leukemia virus. Both ex vivo produced VLPs and plasmid DNA encoding VLPs can be used as vaccines. Here, we report that immunizations with plasmid DNA forming VLPs pseudotyped with HCV E1 and E2 envelope glycoproteins (HCV-specific plasmo-retroVLPs) induce strong T-cell-mediated immune responses that can be optimized by using proper DNA delivery methods and/or genetic adjuvants. Additionally, multigenotype or multi-specific T-cell responses were observed after immunization with plasmids that encode VLPs pseudotyped with E1E2 derived from numerous viral genotypes and/or displaying NS3 antigen in capsid proteins. While homologous prime-boost immunizations with HCV-specific plasmo-retroVLPs or ex vivo produced VLPs induce a low level of specific antibody responses, optimal combination of plasmo-retroVLPs and VLPs was identified for inducing HCV-specific T-cell and B-cell responses as well as neutralizing antibodies. Altogether, these results have important meanings for the development of anti-HCV preventive vaccines and exemplify the flexibility and potential of our retrovirus-based platform in inducing broad cellular and humoral immune responses.     

Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP,a STING activator or archaeosomes

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice.Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4⁺ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.     

Immunoadjuvant efficacy of plasmids with multiple copies of a CpG motif coadministrated with avian influenza vaccine in chickens

Unmethylated CpG motifs are capable of evoking a range of immunostimulatory effects in vertebrates and have tremendous potential to be used as therapeutic agents and adjuvants. This particular type of CpG motif has been demonstrated to be an excellent immune adjuvant mediated by Toll-like receptor 9 (TLR9) in various mammalian vaccines; however, only a few studies confirm its efficacy in avian vaccines. In the present study, immunomodulatory activities of plasmids with various copy numbers of a CpG motif were evaluated in chickens inoculated with an avian influenza vaccine. Results showed that the plasmid with 64 copies of the CpG motif (64CpG-plasmid) significantly enhanced the mRNA expressions of interferon-gamma (IFN-γ), TLR3 and TLR7 in chicken splenocytes compared to plasmids with lesser copies of the CpG motif in vitro. Chickens inoculated with the H5N2 avian influenza inactivated vaccines (V52) coadministrated with the 64CpG-plasmid (V52-64CpG) showed significant increments of hemagglutination inhibition (HI) titers, peripheral blood mononuclear cell (PBMC) proliferation, and mRNA expressions of IFN-α, IFN-γ, TLR3, TLR7 and TLR21 in splenocytes as compared to those of chickens inoculated with V52 alone, V52 adjuvanted with aluminum gel (V52-gel), or with V52-gel plus vector. Additionally, following challenge with a highly virulent H5N1 strain, a higher survival rate (100%) was observed in chickens inoculated with V52-64CpG as compared to those that received V52-gel (80%) or PBS (0%). The 64CpG-plasmid significantly enhanced chicken immunity in vitro and in vivo; thus it can be a potent adjuvant in an avian influenza vaccine for chickens.     

Deletion of N-glycosylation sites of hepatitis C virus envelope protein E1 enhances specific cellular and humoral immune responses

N-linked glycosylations of viral proteins have been implicated in immunogenicity. In this study, the effects of the N-linked glycosylation of the hepatitis C virus (HCV) E1 protein, a naturally poor immunogen, on the induction of specific immune response were examined. We constructed the plasmids containing the genes encoding both wild type and mutant E1 proteins in which N-linked glycosylation sites are mutated individually or in combination by site-directed mutagenesis. The immunogenicity of wild type E1 and six mutated E1 proteins was analyzed in BALB/C mice using a DNA-based vaccination approach. We found that E1-M2 mutant (at site of N209SS) significantly enhanced E1-specific CD8(+)T cells cytotoxic T lymphocytes (CTL) activities, expression of IFN-gamma producing T cells, and suppression of tumor growth. While E1-M4 mutant (at site of N305CS) induced the highest specific antibody response among all groups. Moreover, E1 wild-type vaccinated mice developed a mixture of IgG1 and Ig2a, but E1-M2 mutant induced only IgG2a isotype, and E1-M4 mutant dominantly developed IgG1 isotype. Our data showed that N-linked glycosylation can limit both cellular and antibody response to the HCV E1 protein and deletion of the N-glycosylation sites at N209SS and N305CS of hepatitis C virus envelope protein E1 provided potential applications for the development of DNA vaccine with enhanced immunogenicity.     

我国HCV感染株基因分型及其意义的研究

应用型特异性探针杂交法及型特异性引物法两种ＨＣＶ基因分型方法对我国ＨＣＶ 感染株基因型别构成特点及其意义进行了初步研究。结果表明，１．我国 ＨＣＶ基因型表现为多种型别共存，但以 Ｋ１（１ｂ）型和Ｋ２（２）型为主，混合型比例亦较高；２．结合临床背景分析，Ｋ１型似与活动性肝病关系密切，慢性迁延性肝病及所谓合格献血员以Ｋ２型为主，混合型多与多次受血、透析有关；３．输血－受血配对研究结果提示，受血者ＨＣＶ基因型与所输注污染的血液或血制品ＨＣＶ基因型有良好的一致性，基因分型方法可以作为调查ＨＣＶ传播途径的重要手段。     

Enhanced cellular immunity to hepatitis C virus nonstructural proteins by codelivery of granulocyte macrophage-colony stimulating factor gene in intramuscular DNA immunization

Hepatitis C virus (HCV) nonstructural (NS) proteins appeared to be important targets for HCV vaccine development, since NS-specific T-helper-cell responses are associated with clearance from acute HCV infection. In this report, we have constructed a plasmid, pTV-NS345, that encodes the HCV NS3, NS4 and NS5 proteins (NS345) and a bicistronic plasmid, PTV-NS345/GMCSF, in which the HCV NS345 polyprotein and GMCSF are translated independently. Intramuscular inoculation with pTV-NS345 plasmid DNA into the Buffalo rats generated both antibody and T-cell proliferative responses to each NS protein. The expression of GMCSF, together with HCV NS345 proteins, appeared to significantly increase T-cell proliferative responses. In particular, the inoculation of a bicistronic plasmid generated higher T-cell proliferative responses to each NS protein than did the coinjection of two separate plasmids, pTV-NS345 and pTV-GMCSF. These results demonstrate that the codelivery of GMCSF augmented HCV NS345-specific cellular immunity and that the intensity of the immunity was differed depending on how GMCSF gene is codelivered.