E7 DNA Vaccine Research Articles

Enhanced synergistic antitumor effect of a DNA vaccine with anticancer cytokine, MDA-7/IL-24, and immune checkpoint blockade

BackgroundMDA-7/IL-24 cytokine has shown potent antitumor properties in various types of cancer without exerting any significant toxicity on healthy cells. It has also been proved to encompass pro-immune Th1 cytokine-like behavior. Several E7 DNA vaccines have developed against human papillomavirus (HPV)-related cervical cancer. However, the restricted immunogenicity has limited their clinical applications individually. To address this deficiency, we investigated whether combining the E7 DNA vaccine with MDA-7/IL-24 as an adjuvant would elicit efficient antitumor responses in tumor-bearing mouse models. Next, we evaluated how suppression of immunosuppressive IL-10 cytokine would enhance the outcome of our candidate adjuvant vaccine.MethodsFor this purpose, tumor-bearing mice received either E7 DNA vaccine, MDA-7/IL-24 cytokine or combination of E7 vaccine with MDA-7/IL-24 adjuvant one week after tumor challenge and boosted two times with one-week interval. IL-10 blockade was performed by injection of anti-IL-10 mAb before each immunization. One week after the last immunization, mice were sacrificed and the treatment efficacy was evaluated through immunological and immunohistochemical analysis. Moreover, the condition of tumors was monitored every two days for six weeks intervals from week 2 on, and the tumor volume was measured and compared within different groups.ResultsA highly significant synergistic relationship was observed between the E7 DNA vaccine and the MDA-7/IL-24 cytokine against HPV-16+ cervical cancer models. An increase in proliferation of lymphocytes, cytotoxicity of CD8+ T cells, the level of Th1 cytokines (IFN-γ, TNF-α) and IL-4, the level of apoptotic markers (TRAIL and caspase-9), and a decrease in the level of immunosuppressive IL-10 cytokine, together with the control of tumor growth and the induction of tumor regression, all prove the efficacy of adjuvant E7&IL-24 vaccine when compared to their individual administration. Surprisingly, vaccination with the DNA E7&IL-24 significantly reduced the population of Regulatory T cells (Treg) in the spleen of immunized mice compared to sole administration and control groups. Moreover, IL-10 blockade enhanced the effect of the co-administration by eliciting higher levels of IFN-γ and caspase-9, reducing Il-10 secretion and provoking the regression of tumor size.ConclusionThe synergy between the E7 DNA vaccine and MDA-7/IL-24 suggests that DNA vaccines’ low immunogenicity can be effectively addressed by coupling them with an immunoregulatory agent. Moreover, IL-10 blockade can be considered a complementary treatment to improve the outcome of conventional or novel cancer therapies.

Co-administration of a plasmid encoding CD40 or CD63 enhances the immune responses to a DNA vaccine against bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) causes substantial economic losses in the livestock industry worldwide. Plasmids encoding the BVDV E2 protein are potential DNA vaccines against BVDV, but their immunogenicity has been insufficient. Here, we investigated the adjuvant effect of CD40 and CD63 plasmids on the immune responses to a BVDV E2 DNA vaccine in mice. We constructed pUMVC4a-based plasmids encoding the BVDV E2 protein (pE2), mouse CD40 (pCD40), or mouse CD63 (pCD63). Protein expression by each plasmid was confirmed through Western blot analysis and immunofluorescence staining of cultured cell lines. BALB/c mice were immunized intradermally twice with pE2 in combination with, or without, pCD40 or pCD63, with 3 weeks between the two doses. pE2 with pCD40 induced significantly higher neutralizing antibody titers against BVDV than pE2 alone. pE2 with pCD63 induced significantly higher anti-E2 IgG2a antibody titers than pE2 alone. Furthermore, pE2 with pCD40 or pCD63 induced significantly increased lymphocyte proliferation and interferon (IFN)-γ production in response to BVDV, compared with E2 alone. These results suggest that a plasmid encoding CD40 or CD63 can be used as an adjuvant to enhance immune responses to DNA vaccines against BVDV.

Enhancing immunogenicity of HPV16 E7 DNA vaccine by conjugating codon-optimized GM-CSF to HPV16 E7 DNA

ObjectiveTo generate immunity against human papillomavirus (HPV), the use of a recombinant DNA vaccine to carry an appropriate target gene is a promising and cost-effective approach. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that enhances the efficacy of vaccines by promoting the development and prolongation of humoral and cellular immunity. In this study, we linked codon-optimized GM-CSF (cGM-CSF) to the HPV16 E7 sequence as fused protein and evaluated the immunogenic potential of this DNA vaccine. Materials and methodsWe have demonstrated that cGM-CSF enhanced immunity against tumor challenges by generating and promoting the proliferation of HPV16 E7-specific CD8+ T cells, which secrete IFN-γ in the murine model. In this study, we aimed to evaluate the immunogenic potential of DNA vaccine that constructed by linking codon-optimized GM-CSF to HPV16 E7 sequence in the animal model. We study the half-life of RNA decay and cellular location of HPV16 E7 by Q-PCR and Western blot. We also assess immune response in the animal model by flow cytometry and ELISA. ResultsThe cGM–CSF–E7 sequence increased and extended the expression of E7 mRNA, in comparison with the E7 sequence alone. Mice vaccinated with the cGM–CSF–E7 DNA vaccine exhibited a slower rate of tumor growth than those vaccinated with the unconjugated E7 DNA vaccine. We also found that the CD4 and CD8+ T cells from these mice showed strong secretion of IFN-γ. ConclusionThrough in vivo antibody depletion experiments, we demonstrated that both CD4+ and CD8+ T cells play an important role in the suppression of tumor growth.

EPCL Electrospun Microfibrous Layers for Immune Assays: Sensitive ELISA for the Detection of Serum Antibodies Against HPV16 E7 Oncoprotein.

Human papillomavirus (HPV) type 16 is the etiologic agent of more than 50% anal/cervical cancers and about 20% oropharyngeal cancers. HPV16 E6 and E7 oncogenes favor the transformation and are essential for maintaining the transformed status. Serum anti-E6 and anti-E7 antibodies appear to have prognostic significance for HPV-associated cancers. However, most of the previous attempts to establish diagnostic tools based on serum detection of E6 and/or E7 antibodies have been unsuccessful, mainly due to the low accuracy of applied tests. This paper reports on a feasibility study to prove the possibility to easily immobilize HPV16 E7 onto electrospun substrates for application in diagnostic tools. In this study, poly(ε-caprolactone) electrospun scaffolds (called ePCL) are used to provide a microstructured substrate with a high surface-to-volume ratio, capable of binding E7 proteins when used for enzyme-linked immunosorbent assay (ELISA) tests. ePCL functionalized with E7 exhibited superior properties compared to standard polystyrene plates, increasing the detection signal from serum antibodies by 5-6 times. Analysis of the serum samples from mice immunized with HPV16 E7 DNA vaccine showed higher efficiency of this new anti-E7 ePCL-ELISA test vs control in E7-specific antibody detection. In addition, ePCL-E7-ELISA is prepared with a relatively low amount of antigen, decreasing the manufacturing costs.

Brucella abortus RB51 lipopolysaccharide influence as an adjuvant on the therapeutic efficacy of HPV16 L1 and HPV16 E7 DNA vaccines.

Objective(s):Human papillomavirus (HPV) is a primary contributing agent of cervical cancer. Eradication of HPV-related infections requires therapeutic strategies. We used Brucella abortus RB51 rough lipopolysaccharide (R-LPS) as an adjuvant along with two HPV16 therapeutic DNA vaccines, pcDNA3-E7 and pcDNA3-L1, for improving DNA vaccine efficacy. Materials and Methods:For evaluation of the B. abortus LPS adjuvant efficacy in combination with DNA vaccines to induce cellular immune responses, C57BL/6 mice were immunized with the DNA vaccines, with or without R-LPS adjuvant. IFN-γ and IL-4 cytokines assay was carried out for assessment of cellular and humoral immune responses. Results:Findings indicated that vaccination with pcDNA3-E7 or pcDNA3-L1 alone could induce strong cellular immune responses, but stronger antigen-specific T-cell immune responses were shown by co-administration of HPV16 E7 and HPV16 L1 DNA vaccines along with R-LPS adjuvant. Conclusion:Overall, B. abortus R-LPS through enhancement of T-cell immune responses can be considered an efficient vaccine adjuvant in future studies and trials.

NOX4 Inhibition Potentiates Immunotherapy by Overcoming Cancer-Associated Fibroblast-Mediated CD8 T-cell Exclusion from Tumors.

Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8+ T cells from tumors (not CD4+ T cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8+ T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 "normalized" CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers. SIGNIFICANCE: NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8+ T-cell exclusion. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/9/1846/F1.large.jpg.See related commentary by Hayward, p. 1799.

British Society for Gene and Cell Therapy Annual Conference Wednesday 19th June – Friday 21st June 2019 University of Sheffield, Sheffield, UK www.bsgct.org

British Society for Gene and Cell Therapy Annual Conference Wednesday 19th June – Friday 21st June 2019 University of Sheffield, Sheffield, UK www.bsgct.org

MTOR Inhibitors Can Enhance the Anti-Tumor Effects of DNA Vaccines through Modulating Dendritic Cell Function in the Tumor Microenvironment.

The life span of dendritic cells (DCs) can become short following induced activation, which is associated with metabolic transition due to the regulation of mechanistic target of rapamycin (mTOR). The purpose of this study was to investigate the potential of inhibiting mTOR to modulate DC functions for elevating the anti-tumor effects of DNA vaccines. Therefore, the influences of various inhibitors of mTOR (mTORi) on the expressions of DC maturation markers, the abilities of antigen presenting and processing of BMM-derived DCs and the tumor killing effects of E7-specific CD8+ T lymphocytes activated by BMM-derived DCs were in vitro examined. The anti-tumor effects of connective tissue growth factor (CTGF)/E7 DNA vaccine and/or mTORi were also in vivo analyzed. In our study, suppressive effects of mTORi on the DC maturation markers expressed on BMMCs could be reversed. The mTORi-treated mature BMM-derived DCs tended to be non-apoptotic. These mTORi-treated BMM-derived DCs could have better antigen presenting and processing abilities. The E7-specific cytotoxic CD8+ T lymphocytes could have more potent tumoricidal activity following activation of mTORi-treated BMM-derived DCs. For tumor-bearing mice, those treated with CTGF/E7 DNA vaccine and mTORi indeed can have higher percentages of mature DCs in the TME, better disease control and longer survivals. Consequently, application of mTORi can be a pharmacological approach for temporally increasing life span, antigen presenting and antigen processing of DCs to strengthen the therapeutic outcome of cancer immunotherapy.

A DNA Vaccine Encoding SA-4-1BBL Fused to HPV-16 E7 Antigen Has Prophylactic and Therapeutic Efficacy in a Cervical Cancer Mouse Model.

The SA-4-1BBL, an oligomeric novel form of the natural ligand for the 4-1BB co-stimulatory receptor of the tumor necrosis factor (TNF) superfamily, as a recombinant protein has potent pleiotropic effects on cells of innate, adaptive, and regulatory immunity with demonstrated therapeutic efficacy in several tumor models. However, the production of soluble form of SA-4-1BBL protein and quality control is time and resource intensive and face various issues pertinent to clinical development of biologics. The present study sought to take advantage of the simplicity and translatability of DNA-based vaccines for the production and delivery of SA-4-1BBL for cancer immune prevention and therapy. A chimeric HPV-16 E7 DNA vaccine (SP-SA-E7-4-1BBL) was constructed that contains the signal peptide (SP) of calreticulin (CRT), streptavidin (SA) domain of SA-4-1BBL, HPV-16 E7 double mutant gene, and the extracellular domain of mouse 4-1BBL. Immunization by gene gun with SP-SA-E7-4-1BBL induced greater prophylactic as well as therapeutic effects in C57BL/6 mice against TC-1 tumor model compared with immunization with E7wt, SP-SA-4-1BBL or reference-positive control CRT-E7wt. The therapeutic efficacy of the DNA vaccine was associated with increased frequency of E7-specific T cells producing interferon (IFN)-γ. Overall, our data suggest that this DNA-based vaccine strategy might represent a translational approach because it provides a simpler and versatile alternative to a subunit vaccine based on SA-4-1BBL and E7 proteins.

Antitumor Immunity Induced by Genetic Immunization with Chitosan Nanoparticle Formulated Adjuvanted for HPV-16 E7 DNA Vaccine.

In recent years attention has been paid to develop effective adjuvant systems for DNA vaccines. Co-formulation of a gene delivery vector with an immunostimulator can enhance therapeutic efficiency of DNA vaccine. To investigate the efficacy of chitosan as a nanodelivery system to enhance antitumor effects of human papilloma virus (HPV)-16 DNA vaccine with IL-12 gene for protection against TC-1 tumor using an animal model. The mice were challenged by subcutaneous injection of TC-1 cells and immunized intramuscularly with DNA vaccine thrice at seven-day intervals. One week after the last immunization, mice were sacrificed and antitumor effects were assessed through measuring lymphocyte proliferation, cytotoxicity, cytokines production, and tumor regression. We found that co-formulation and co-administration of chitosan nanoparticles and IL-12 with HPV-16 E7 DNA vaccine induced higher antitumor effects compared with chitosan or IL-12 alone. E7-specific lymphocyte proliferation index and CTL activity were found to be significantly higher in combination group in comparison to single vaccination with either chitosan or IL-12. Co-formulation of chitosan and IL-12 resulted in higher IFN-γ and IL-4, and decreased IL-10 production. Furthermore, combined vaccination highly inhibited the tumor progression compared with chitosan or IL-12 alone. Chitosan nanoparticle is a promising delivery system for DNA vaccine and IL-12 is an effective genetic adjuvant for the induction of strong antitumor immune response.

Enhanced immune responses to E2 protein and DNA formulated with ISA 61 VG administered as a DNA prime–protein boost regimen against bovine viral diarrhea virus

The aim of this study was to develop and test an optimal vaccination strategy against bovine viral diarrhea virus (BVDV) based on the E2 glycoprotein of the BJ1305 strain. To achieve higher E2-specific antibody titers and to broaden the cellular immune response, a plasmid encoding the E2 protein (pcDNA3.1-E2) was constructed and a purified recombinant E2 protein was generated. The E2 protein was emulsified in the adjuvant ISA 61 VG prior to administration. We immunized mice three times with pcDNA3.1-E2 or the recombinant E2 protein or primed twice with pcDNA3.1-E2 and boosted once with the E2 protein. To evaluate the protection against BVDV conferred by the vaccines, the mice were challenged with BVDV strain Oregon C24V after the third immunization. Although all immunized mice developed humoral and cellular immune responses, the E2-specific antibody titers in the DNA prime–protein boost group were significantly higher than those elicited by either the DNA or the protein vaccine. In addition, vaccination with the E2 DNA vaccine induced higher percentages of CD4+IFN-γ+ T cells and CD8+IFN-γ+ T cells among total CD3+ T cells than the other regimens. The predominant antibody subclass in the vaccinated mice was IgG1. Serum tumor necrosis factor alpha (TNF-α) levels in the DNA prime–protein boost group were significantly higher after the third immunization than in the other groups. Moreover, the mice treated with the DNA prime–protein boost vaccination regimen acquired protection against BVDV challenge, as shown by a significant reduction of viremia, only minor pathological changes, and a lower viral antigen burden than in the control and solo vaccinated mice. These results demonstrate the potential advantage of a DNA prime–protein boost vaccination approach over a solo vaccination for the prevention of BVDV. The ability of this vaccine strategy to control and eradicate BVD in herds warrants further investigation.

62P - Evaluation the effect of Brucella abortus RB51 lipopolysaccharide as an adjuvant on the efficiency of HPV-16 L1 and HPV-16 E7 DNA vaccines in C57BL/6 mice

62P - Evaluation the effect of Brucella abortus RB51 lipopolysaccharide as an adjuvant on the efficiency of HPV-16 L1 and HPV-16 E7 DNA vaccines in C57BL/6 mice

Enhancement of therapeutic DNA vaccine potency by melatonin through inhibiting VEGF expression and induction of antitumor immunity mediated by CD8+ T cells.

To be effective, therapeutic cancer vaccines should stimulate both an effective cell-mediated and a robust cytotoxic CD8+T-cell response against human papillomavirus (HPV)-infected cells to treat the pre-existing tumors and prevent potential future tumors. In this study, the therapeutic experiments were designed in order to evaluate antitumor effect against the syngeneic TC-1 tumor model. The anti-tumorefficacy of a HPV-16 E7 DNA vaccine adjuvanted with melatonin (MLT) was evaluated in a C57BL/6 mousetumormodel by measuringtumorgrowth post vaccination and the survival rate oftumor-bearingmice, analyzing the specific lymphocyte proliferation responses in control and vaccinatedmiceby MTT assay. TheE7-specific cytotoxic T cells (CTL) were analyzed bylymphocyteproliferation and lactate dehydrogenates (LDH) release assays. IFN-γ, IL-4 and TNF-α secretion in splenocyte cultures as well as vascular endothelial growth factor (VEGF) and IL-10 in the tumor microenvironment were assayed by ELISA. Our results demonstrated that subcutaneous administration of C57BL/6micewith a DNA vaccine adjuvanted with MLT dose-dependently and significantly induced strong HPV16 E7-specific CD8+ cytotoxicity and IFN-γ and TNF-α responses capable of reducing HPV-16 E7-expressing tumor volume. A significantly higher level of E7-specific T-cell proliferation was also found in the adjuvanted vaccine group. Furthermore, tumor growth was significantly inhibited when the DNA vaccine was combined with MLT and the survival time of TC-1 tumor bearing mice was also significantly prolonged. In vivo studies further demonstrated that MLT decreased the accumulation of IL-10 and VEGF in the tumor microenvironment of vaccinated mice. These data indicate that melatonin as an adjuvant augmented the cancer vaccine efficiency against HPV-associated tumors in a dose dependent manner.

Combination of the toll like receptor agonist and α-Galactosylceramide as an efficient adjuvant for cancer vaccine.

BackgroundDNA vaccines have emerged as an attractive approach for the generation of cytotoxic T lymphocytes (CTL). In our previous study, we found That Toll like receptor (TLR) ligands are promising candidates for the development of novel adjuvants for DNA vaccine. To improve the efficacy of DNA vaccine directed against human papillomavirus (HPV) tumors, we evaluated whether co-administration of a TLR4 ligand, monophosphoryl lipid A (MPL), and Natural Killer T Cell Ligand α-Galactosylceramide(α-GalCer) adjuvants with DNA vaccine would influence the anti-tumor efficacy of DNA vaccinations.MethodsWe investigated the effectiveness of α-GalCer and MPL combination as an adjuvant with an HPV-16 E7 DNA vaccine to enhance antitumor immune responses.ResultsBy using adjuvant combination for a DNA vaccine, we found that the levels of lymphocyte proliferation, CTL activity, IFN- γ, IL-4 and IL-12 responses, and tumor protection against TC-1 cells were significantly increased compared to the DNA vaccine with individual adjuvants.In addition, inhibition of IL-18 signaling during vaccination decreased IFN-γ responses and tumor protection, and that this inhibition suggested stimulatory role of IL-18 in adjuvant effects of α-GalCer and MPL combination.ConclusionThe strong adjuvanticity associated with α-GalCer/MPL combination may to be an important tool in the development of novel and strong cancer immunotherapy.

Enhancement of immune response to a hepatitis C virus E2 DNA vaccine by an immunoglobulin Fc fusion tag

Neutralizing antibodies and cellular immune response both play essential roles in the clearance of Hepatitis C virus (HCV) infection. The envelope glycoprotein E2 is a major target for producing neutralizing antibodies against HCV. Here, we constructed a recombinant plasmid, termed pcDNA3.1-E2-Fc, to express HCV E2 with an immunoglobulin Fc fusion tag (E2-Fc). Importantly, we found that the titers of E2-specific IgG from mice immunized with pcDNA3.1-E2-Fc were significantly higher than that from mice immunized with pcDNA3.1-E2. Moreover, pcDNA3.1-E2-Fc immunization could boost E2-specific lymphocyte proliferation and enhance the secretion of IFN-γ by lymphocytes upon in vitro stimulation with soluble E2 compared to pcDNA3.1-E2 immunization. Neutralization assays showed that serum from pcDNA3.1-E2-Fc immunized mice exhibited more effective neutralizing capacity of HCVpp entry into Huh-7 cells compared with that from pcDNA3.1-E2 immunized mice, although both of the sera could inhibit the virus entry. Taken together, our results imply that pcDNA3.1-E2-Fc immunization could enhance E2-specific humoral and cellular immune response in mice and thus provide a promising candidate for the development of an HCV vaccine.

Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems.

BackgroundCervical cancer is the second-most-common cause of malignancies in women worldwide, and the oncogenic activity of the human papilloma virus types (HPV) E7 protein has a crucial role in anogenital tumors. In this study, we have designed a therapeutic vaccine based on chitosan nanodelivery systems to deliver HPV-16 E7 DNA vaccine, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer. We have developed a Nano-chitosan (NCS) as a carrier system for intramuscular administration using a recombinant DNA vaccine expressing HPV-16 E7 (NCS-DNA E7 vaccine). NCS were characterized in vitro for their gene transfection ability.ResultsThe transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. In addition, NCS-DNA E7 vaccine induced the strongest E7-specific CD8+ T cell and interferon γ responses in C57BL/6 mice. Mice vaccinated with NCS-DNA E7 vaccine were able to generate potent protective and therapeutic antitumor effects against challenge with E7-expressing tumor cell line, TC-1.ConclusionsThe strong therapeutic effect induced by the Chitosan-based nanodelivery suggest that nanoparticles may be an efficient carrier to improve the immunogenicity of DNA vaccination upon intramuscular administration and the platform could be further exploited as a potential cancer vaccine candidate in humans.

DNA vaccine encoding HPV-16 E7 with mutation in L-Y-C-Y-E pRb-binding motif induces potent anti-tumor responses in mice

Cervical cancer is the second most common cancer among women worldwide and remains a clinical problem despite improvements in early detection and therapy. The human papillomavirus (HPV) type 16 (HPV16) E7 oncoprotein expressed in cervical carcinoma cells are considered as attractive tumor-specific antigen targets for immunotherapy. Since the transformation potential of the oncogenes, vaccination based of these oncogenes is not safe. In present study, DNA vaccine expressing the modified variant with mutation in pRb-binding motif of the HPV-16 E7 oncoprotein was generated.A novel modified E7 gene with mutation in LYCYE motif was designed and constructed and the immunogenicity and antitumor effect of therapeutic DNA vaccines encoding the mutant and wild type of E7 gene were investigated. The L-Y-C-Y-E pRb-binding motif of E7 proteins has been involved in the immortalization and transformation of the host cell.The results showed that the mutant and wild type HPV-16 E7 vectors expressed the desired protein. Furthermore, the immunological mechanism behind mutant E7 DNA vaccine can be attributed at least partially to increased cytotoxic T lymphocyte, accompanied by the up-regulation of Th1-cytokine IFN-γ and TNF-β and down-regulation of Th3-cytokine TGF-β. Immunized mice with mutant plasmid demonstrated significantly stronger cell immune responses and higher levels of tumor protection than wild-type E7 DNA vaccine.The results exhibit that modified E7 DNA vaccine may be a promising candidate for development of therapeutic vaccine against HPV-16 cancers.

Comparing the effect of Toll-like receptor agonist adjuvants on the efficiency of a DNA vaccine

We have investigated whether poly(I:C) Toll-like receptor 3 (TLR3) and resiquimod Toll-like receptor 7 (TLR7) agonists can serve as vaccine adjuvants and promote the efficiency of therapeutic DNA vaccination against tumors expressing the human papilloma virus 16 (HPV-16) E7 protein. For this purpose, C57BL/6 mice were inoculated with 2 × 10(5) TC-1 cells, and they were then immunized with HPV-16 E7 DNA vaccine alone or with 50 μg of resiquimod or poly(I:C) individually. We found that poly(I:C) and resiquimod could induce more antigen-specific lymphocyte proliferation and cytolytic activity compared to vaccination with E7 DNA alone. While E7 DNA had no significant inhibitory effect on tumor growth, co-administration of poly(I:C) and resiquimod with E7 DNA induced significant tumor regression. Peripheral and local cytokine assays demonstrated that co-administration of poly(I:C) and resiquimod with E7 DNA induced circulating antigen-specific IFN-γ and nonspecific intratumoral IL-12. TLR3 and TLR7 agonists can be used to enhance the immune response to DNA vaccine immunogens. Taken together, these data indicate that combined vaccination with DNA encoding HPV-16 E7 plus TLR agonists provides a strategy for improving the efficacy of a vaccine as a possible immunotherapeutic strategy for 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.

Two Doses of Bovine Viral Diarrhea Virus DNA Vaccine Delivered by Electroporation Induce Long-Term Protective Immune Responses

Bovine viral diarrhea virus (BVDV) is a pathogen of major importance in cattle, so there is a need for new effective vaccines. DNA vaccines induce balanced immune responses and are relatively inexpensive and thus promising for both human and veterinary applications. In this study, newborn calves with maternal antibodies were vaccinated intramuscularly (i.m.) with a BVDV E2 DNA vaccine with the TriGrid Delivery System for i.m. delivery (TDS-IM). Two doses of this vaccine spaced 6 or 12 weeks apart were sufficient to induce significant virus-neutralizing antibody titers, numbers of activated T cells, and reduction in viral shedding and clinical presentations after BVDV-2 challenge. In contrast to the placebo-treated animals, the vaccinated calves did not lose any weight, which is an excellent indicator of the well-being of an animal and has a significant economic impact. Furthermore, the interval between the two vaccinations did not influence the magnitude of the immune responses or degree of clinical protection, and a third immunization was not necessary or beneficial. Since electroporation may enhance not only the magnitude but also the duration of immunity after DNA immunization, the interval between vaccination and challenge was extended in a second trial, which showed that two doses of this E2 DNA vaccine again significantly reduced clinical disease against BVDV for several months. These results are promising and support this technology for use against infectious diseases in cattle and large species, including humans, in general.