Antitumor therapeutic effects of e7 subunit and DNA vaccines in an animal cervical cancer model: antitumor efficacy of e7 therapeutic vaccines is dependent on tumor sizes, vaccine doses, and vaccine delivery routes

We previously reported that E7 subunit and DNA vaccines are both capable of inducing antitumor protection through induction of antigen-specific CTL. In this study, we investigated their ability to control established tumors according to tumor size, vaccine doses, and vaccine delivery routes. Antitumor therapeutic efficacy of both vaccine types was dependent on tumor burden. However, E7 subunit vaccines induced a higher level of antitumor therapeutic activities at the tested dose compared to DNA vaccines. This was concomitant with induction of antibody, CTL, and IFN-gamma responses, as well as histologic changes (heavy infiltration of lymphocytes and presence of apoptotic bodies). In vaccine dose titration assays, 50 and 100 microg of DNA vaccines exhibited an equivalent antitumor efficacy to 0.5 and 1 microg of E7 subunit vaccines, respectively, i.e., a 100-fold difference in E7 dosage, suggesting the importance of vaccine doses for achieving antitumor immunity. Furthermore, tumors of a larger size were controlled by intratumoral injection with E7 subunit vaccines, underscoring the importance of vaccine delivery routes for antitumor therapeutic efficacy. Thus, these data suggest that antitumor therapeutic efficacy of E7 therapeutic vaccines is determined by vaccine doses, vaccine delivery routes, and tumor sizes, and that these vaccines could be another addition to conventional therapy modalities against cervical cancer.     

Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer.     

Repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes and improved protective antitumor effects

The use of DNA vaccines for generating antigen-specific CD8+ T cell responses has been well established. However, little is known about the quantitative and qualitative aspects of CD8+ T cell responses and protective immunity generated after repeated DNA vaccinations. We used human papillomavirus (HPV) type-16 E7 as a model tumor antigen in an E7-expressing tumor model, TC-1, to assess the influence of the frequency of DNA vaccinations on E7-specific immunological and antitumor responses. Mice were vaccinated with 1–4 inoculations of pcDNA3-E7 DNA. Immunological assays and tumor protection experiments were performed to assess the effect of repeated E7 DNA vaccination on E7-specific T cells and E7-expressing tumors. Our results demonstrated that mice receiving an increased number of E7 DNA vaccinations exhibited higher E7-specific CTL activity, a rapid expansion of E7-specific IFN--secreting CD8+ T cells upon stimulation with E7 antigen, and a stronger antitumor effect against an E7-expressing tumor. Furthermore, we found that increasing the number of E7 DNA vaccinations followed by vaccinia booster enhanced the functional avidity of E7-specific CD8+ T cells. Our data suggest that quantitative and qualitative characteristics of antigen-specific CD8+ T cell responses and the ensuing protective antitumor effect can be influenced by the frequency of DNA vaccinations. These results have important clinical implications for the use of naked DNA vaccines in cancer immunotherapy.     

Enhancement of DNA vaccine potency through linkage of antigen gene to ER chaperone molecules,ER-60, tapasin,and calnexin

DNA vaccines have emerged as an attractive approach for generating antigen-specific immunotherapy. Strategies that enhance antigen presentation may potentially be used to enhance DNA vaccine potency. Previous experiments showed that chimeric DNA vaccines utilizing endoplasmic reticulum (ER) chaperone molecules, such as Calreticulin (CRT), linked to an antigen were capable of generating antigen-specific CD8⁺ T cell immune responses in vaccinated mice. In this study, we tested DNA vaccines encoding the ER chaperone molecules ER-60, tapasin (Tap), or calnexin (Cal), linked to human papillomavirus type 16 (HPV-16) E7 for their abilities to generate E7-specific T cell-mediated immune responses and antitumor effects in vaccinated mice. Our results demonstrated that vaccination with DNA encoding any of these chaperone molecules linked to E7 led to a significant increase in the frequency of E7-specific CD8⁺ T cell precursors and generated stronger antitumor effects against an E7-expressing tumor in vaccinated mice compared to vaccination with wild-type E7 DNA. Our data suggest that DNA vaccines employing these ER chaperone molecules linked to antigen may enhance antigen-specific CD8⁺ T cell immune responses, resulting in a significantly more potent DNA vaccine.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

The potential of DNA vaccination against tumor-associated antigens for antitumor therapy

Conventional treatment approaches for malignant tumors are highly invasive and sometimes have only a palliative effect. Therefore, there is an increasing demand to develop novel, more efficient treatment options. Increased efforts have been made to apply immunomodulatory strategies in antitumor treatment. In recent years, immunizations with naked plasmid DNA encoding tumor-associated antigens have revealed a number of advantages. By DNA vaccination, antigen-specific cellular as well as humoral immune responses can be generated. The induction of specific immune responses directed against antigens expressed in tumor cells and displayed e.g., by MHC class I complexes can inhibit tumor growth and lead to tumor rejection. The improvement of vaccine efficacy has become a critical goal in the development of DNA vaccination as antitumor therapy. The use of different DNA delivery techniques and coadministration of adjuvants including cytokine genes may influence the pattern of specific immune responses induced. This brief review describes recent developments to optimize DNA vaccination against tumor-associated antigens. The prerequisite for a successful antitumor vaccination is breaking tolerance to tumor-associated antigens, which represent "self-antigens." Currently, immunization with xenogeneic DNA to induce immune responses against self-molecules is under intensive investigation. Tumor cells can develop immune escape mechanisms by generation of antigen loss variants, therefore, it may be necessary that DNA vaccines contain more than one tumor antigen. Polyimmunization with a mixture of tumor-associated antigen genes may have a synergistic effect in tumor treatment. The identification of tumor antigens that may serve as targets for DNA immunization has proceeded rapidly. Preclinical studies in animal models are promising that DNA immunization is a potent strategy for mediating antitumor effects in vivo. Thus, DNA vaccines may offer a novel treatment for tumor patients. DNA vaccines may also be useful in the prevention of tumors with genetic predisposition. By DNA vaccination preventing infections, the development of viral-induced tumors may be avoided.     

Cancer Immunotherapy Employing an Innovative Strategy to Enhance CD4+ T Cell Help in the Tumor Microenvironment

Chemotherapy and/or radiation therapy are widely used as cancer treatments, but the antitumor effects they produce can be enhanced when combined with immunotherapies. Chemotherapy kills tumor cells, but it also releases tumor antigen and allows the cross-presentation of the tumor antigen to trigger antigen-specific cell-mediated immune responses. Promoting CD4+ T helper cell immune responses can be used to enhance the cross-presentation of the tumor antigen following chemotherapy. The pan HLA-DR binding epitope (PADRE peptide) is capable of generating antigen-specific CD4+ T cells that bind various MHC class II molecules with high affinity and has been widely used in conjunction with vaccines to improve their potency by enhancing CD4+ T cell responses. Here, we investigated whether intratumoral injection of PADRE and the adjuvant CpG into HPV16 E7-expressing TC-1 tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and PADRE was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and PADRE also enhanced the generation of PADRE-specific CD4+ T cells and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci. The treatment regimen presented here represents a universal approach to cancer control.     

A combination of intradermal jet-injection and electroporation overcomes in vivo dose restriction of DNA vaccines

ABSTRACT: BACKGROUND: The use of optimized delivery devices has been shown to enhance the potency of DNA vaccines. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans. METHODS: Herein we evaluated antigen expression and immunogenicity after intradermal delivery of different doses of DNA vaccines by needle or by the Biojector jet-injection device, with or without the addition of electroporation (EP). RESULTS: Neither needle injection augmented by EP nor Biojector alone could induce higher magnitudes of immune responses after immunizations with a high dose of a DNA vaccine as compared to immunizations with a considerably lower dose. Biojector delivery followed by EP, however, overcame this observed dose restriction and induced significantly higher cellular and humoral immune responses after immunization with a high dose of DNA. Furthermore, a close correlation between in vivo antigen expression and cell-mediated immune responses was observed. CONCLUSIONS: These results show that two optimized DNA vaccine delivery devices can act together to overcome dose restrictions of plasmid DNA vaccines.     

Spontaneous elicitation of potent antitumor immunity and eradication of established tumors by administration of DNA encoding soluble transforming growth factor-β II receptor without active antigen-sensitization

Since immunity is generally suppressed by immunoregulatory factors, such as transforming growth factor-beta (TGF-β), interleukin (IL)-10, and vascular endothelial growth factor (VEGF), produced by tumor cells or stromal cells surrounding tumor cells, various kinds of cancer immunotherapy mostly fail to elicit potent antitumor immunity. Herein, we tested whether neutralization of TGF-β can elicit strong antitumor immune responses and tumor regression in tumor-bearing mice. A plasmid DNA, pcDNA-sTGFβR/huIg, encoding a fusion protein consisting of the extracellular domain of TGF-β type II receptor (TGFβRII) and the Fc portion of human IgG heavy chain, was injected through different routes into B6 mice carrying established tumors of E.G7 cells, which consist of the poorly immunogenic tumor cells EL4, transfected with the ovalbumin (OVA) gene. The frequency of OVA-specific cytotoxic T lymphocytes (CTL), in the treated mice. increased resulting in the tumor eradication and relapse-free survival in around 70% of the E.G7-bearing mice. In contrast, administration of mock DNA into E.G7-bearing mice did not elicit tumor-specific immune responses. Therefore, administration of DNA encoding TGFβRII allowed tumor-bearing hosts to elicit sufficiently potent antitumor immune responses without requirement of further active antigen-immunization. This strategy seems to be applicable to clinical therapy against cancer, because it is low-cost, safe, and easy to manipulate.     

Vascular disrupting agent DMXAA enhances the antitumor effects generated by therapeutic HPV DNA vaccines

Antigen-specific immunotherapy using DNA vaccines has emerged as an attractive approach for the control of tumors. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 DNA vaccination to enhance the antitumor effects and E7-specific CD8+ T cell immune responses in treated mice. We determined that treatment with DMXAA generates significant therapeutic effects against TC-1 tumors but does not enhance the antigen-specific immune responses in tumor bearing mice. We then found that combination of DMXAA treatment with E7 DNA vaccination generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes of tumor bearing mice. Furthermore, the DMXAA-mediated enhancement or suppression of E7-specific CD8+ T cell immune responses generated by CRT/E7 DNA vaccination was found to be dependent on the time of administration of DMXAA and was also applicable to other antigen-specific vaccines. In addition, we determined that inducible nitric oxide synthase (iNOS) plays a role in the immune suppression caused by DMXAA administration before DNA vaccination. Our study has significant implications for future clinical translation.     

Induction of specific human primary immune responses to a Semliki Forest virus–based tumor vaccine in a Trimera mouse model

Recombinant Semliki Forest virus (rSFV) enables high-level, transient expression of heterologous proteins in vivo, and is believed to be a superior vector for genetic vaccination, compared with the conventional DNA plasmid. Nonetheless, the efficacy of rSFV-based vaccine in eliciting human immune responses has not been tested. We used a Trimera mouse model, consisting of lethally irradiated BALB/c host reconstituted with nonobese diabetes/severe combined immunodeficiency (NOD/SCID) bone marrow plus human peripheral blood mononuclear cells (PBMCs), to characterize the in vivo immune responses against rSFV-encoded human melanoma antigen MAGE-3. MAGE-3–specific antibody and cytotoxic T lymphocyte (CTL) activity were detected by ELISA and ⁵¹Cr-release assay, respectively, and the responses were compared with those induced by a plasmid DNA vaccine encoding the same antigen. The results showed that rSFV vaccine could elicit human MAGE-3–specific antibody and CTL response in the Trimera mice, and the antitumor responses were more potent than those by plasmid DNA vaccination. This is the first report to evaluate human immune responses to an rSFV-based tumor vaccine in the Trimera mouse model. Our data suggest that rSFV vector is better than DNA plasmid in inducing protective immunity, and the Trimera model may serve as a general tool to evaluate the efficacy of tumor vaccines in eliciting human primary immune response in vivo.     

Delivery of protein antigens to the immune system by fusion-active virosomes: a comparison with liposomes and ISCOMs

The induction of effective cellular and humoral immune responses against protein antigens is of major importance in vaccination strategies against infectious diseases and cancer. Immunization with protein alone in general does not result in efficient induction of cytotoxic T lymphocyte (CTL) and antibody responses. Numerous other immunization strategies have been explored. In this review we will discuss a number of lipid-based antigen delivery systems suitable for the induction of CTL responses. These systems comprise reconstituted virus envelopes (virosomes), liposomes, and immune-stimulating complexes (ISCOMs). We will concentrate on delivery of the protein antigen ovalbumin (OVA) since extensive studies with this antigen have been performed for all of the systems discussed, allowing direct comparison of antigen delivery efficiency. Stimulation of CTL activity requires processing of the antigen in the cytosol of antigen-presenting cells (APCs) and presentation of antigenic peptides on surface major histocompatibility class I complexes (MHC class I). In vitro, the ability of antigen delivery systems to induce MHC class I presentation indeed correlates with their capacity to deliver antigen to the cytosol of cells. This capacity appears to be less important for the induction of cytotoxic T lymphocytes in vivo. Instead, other properties of the antigen delivery system like activation of APCs and induction of T helper cells play a more prominent role. Fusion-active virosomes appear to be a very potent system for induction of CTL activity, most likely since virosomes combine efficient delivery of antigen with general stimulation of the immune system.     

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.     

Fusion of CTLA-4 with HPV16 E7 and E6 Enhanced the Potency of Therapeutic HPV DNA Vaccine

Preventive anti-HPV vaccines are effective against HPV infection but not against existing HPV-associated diseases, including cervical cancer and other malignant diseases. Therefore, the development of therapeutic vaccines is urgently needed. To improve anti-tumor effects of therapeutic vaccine, we fused cytotoxic T-lymphocyte antigen 4 (CTLA-4) with HPV16 E7 and E6 as a fusion therapeutic DNA vaccine (pCTLA4-E7E6). pCTLA4-E7E6 induced significantly higher anti-E7E6 specific antibodies and relatively stronger specific CTL responses than the nonfusion DNA vaccine pE7E6 in C57BL/6 mice bearing with TC-1 tumors. pCTLA4-E7E6 showed relatively stronger anti-tumor effects than pE7E6 in therapeutic immunization. These results suggest that fusing CTLA-4 with E7E6 is a useful strategy to develop therapeutic HPV DNA vaccines. In addition, fusing the C-terminal of E7 with the N-terminal of E6 impaired the functions of both E7 and E6.     

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.     

A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.     

Accelerated immune response to DNA vaccines

DNA vaccines offer considerable promise for improvement over conventional vaccines. For the crucial step of delivering DNA vaccines intracellularly, electroporation (EP) has proven to be highly effective. This method has yielded powerful humoral and cellular responses in various species, including nonhuman primates. In an attempt to further improve DNA vaccination we used micron-size gold particles (which do not bind or adsorb DNA) as a particulate adjuvant which was coinjected with DNA intramuscularly into mice, followed by EP of the target site. The presence of gold particles accelerated the antibody response significantly. Maximum titers against hepatitis B surface antigen (HBsAg) were reached after one boost in 6 weeks, whereas 8 weeks were required without particles. These immunizations were effective in protecting mice against tumor challenge with cancer cells expressing HBsAg as a surrogate cancer antigen. Computer modeling of electric fields and gene expression studies indicate that gold particles do not stimulate EP and subsequent antigen expression. The particles may act as an attractant for immune cells, especially antigen presenting cells. We conclude that particulate adjuvants combined with DNA vaccine delivery by EP reduces the immune response time and may increase vaccine efficacy. This method may become valuable for developing prophylactic as well as therapeutic vaccines. The rapid response may be of particular interest in countering bio-terrorism.     

In Vivo Electroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

DNA vaccines offer advantage over conventional vaccines, as they are safer to use, easier to produce, and able to induce humoral as well cellular immune responses. Unfortunately, no DNA vaccines have been licensed for human use for the difficulties in developing an efficient and safe in vivo gene delivery system. In vivo electroporation (EP)-based DNA delivery has attracted great attention for its potency to enhance cellular uptake of DNA vaccines and function as an adjuvant. Minicircle DNA (a new form of DNA containing only a gene expression cassette and lacking a backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery in terms of improving the levels and the duration of transgene expression in vivo. In this study, as a novel vaccine delivery system, we combined in vivo EP and the minicircle DNA carrying a codon-optimized HIV-1 gag gene (minicircle-gag) to evaluate the immunogenicity of this system. We found that minicircle-gag conferred persistent and high levels of gag expression in vitro and in vivo. The use of EP delivery further increased minicircle-based gene expression. Moreover, when delivered by EP, minicircle-gag vaccination elicited a 2- to 3-fold increase in cellular immune response and a 1.5- to 3-fold augmentation of humoral immune responses compared with those elicited by a pVAX1-gag positive control. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, upregulating inflammatory genes, and recruiting immune cells. Collectively, in vivo EP of minicircle DNA functions as a novel vaccine platform that can enhance efficacy and immunogenicity of DNA vaccines.     

Revealing the potential of DNA-based vaccination: lessons learned from the hepatitis B virus surface antigen

DNA-based vaccination is a novel technique to efficiently stimulate humoral (antibody) and cellular (T cell) immune responses to protein antigens. In DNA-based vaccination, immunogenic proteins are expressed in in vivo transfected cells of the vaccine recipients in their native conformation with correct posttranslational modifications from antigen-encoding expression plasmid DNA. This ensures the integrity of antibody-defined epitopes and supports the generation of protective (neutralizing) antibody titers. Plasmid DNA vaccination is furthermore an exceptionally potent strategy to stimulate CD8+ cytotoxic T lymphocyte (CTL) responses because antigenic peptides are efficiently generated by endogenous processing of intracellular protein antigens. These key features make DNA-based immunization an attractive strategy for prophylactic and therapeutic vaccination against extra- and intracellular pathogens. In this brief review, we summarize the current state of expression vector design, DNA delivery strategies, priming immune responses to intracellular or secreted antigens by DNA vaccines and unique advantages of DNA- versus recombinant protein-based vaccines using the hepatitis B surface antigen (HBsAg) as a model antigen.     

Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte activity by a T-helper cell-independent mechanism

Immunostimulatory DNA sequences (ISS) contain unmethylated CpG dinucleotides within a defined motif. Immunization with ISS-based vaccines has been shown to induce high antigen-specific cytotoxic lymphocyte (CTL) activity and a Th1-biased immune response. We have developed a novel ISS-based vaccine composed of ovalbumin (OVA) chemically conjugated to ISS-oligodeoxynucleotide (ODN). Protein-ISS conjugate (PIC) is more potent in priming CTL activity and Th1-biased immunity than other ISS-based vaccines. Cytotoxic lymphocyte activation by ISS-ODN-based vaccines is preserved in both CD4-/- and MHC class II-/- gene-deficient animals. Furthermore, PIC provides protection against a lethal burden of OVA-expressing tumor cells in a CD8+ cell-dependent manner. These results demonstrate that PIC acts through two unique mechanisms: T-helper-independent activation of CTL and facilitation of exogenous antigen presentation on MHC class I. This technology may have clinical applications in cancer therapy and in stimulating host defense in AIDS and chronic immunosuppression.