Abstract
Genetic immunization strategies have largely focused on the use of plasmid DNA with a gene gun. However, there remains a clear need to further improve the efficiency, safety, and cost of potential DNA vaccines. The gold particle-coated DNA format delivered through a gene gun is expensive, time and process consuming, and raises aseptic safety concerns. This study aims to determine whether a low-pressured gene gun can deliver noncarrier naked DNA vaccine without any particle coating, and generate similarly strong antigen-specific immunologic responses and potent antitumor effects compared with gold particle-coated DNA vaccine. Our results show that mice vaccinated with noncarrier naked chimeric CRT/E7 DNA lead to dramatic increases in the numbers of E7-specific CD8+ T-cell precursors and markedly raised titers of E7-specific antibodies. Furthermore, noncarrier naked CRT/E7 DNA vaccine generated potent antitumor effects against subcutaneous E7-expressing tumors and pre-established E7-expressing metastatic pulmonary tumors. In addition, mice immunized with noncarrier naked CRT/E7 DNA vaccine had significantly less burning effects on the skin compared with those vaccinated with gold particle-coated CRT/E7 DNA vaccine. We conclude that noncarrier naked CRT/E7 DNA vaccine delivered with a low-pressured gene gun can generate similarly potent immunologic responses and effective antitumor effects has fewer side effects, and is more convenient than conventional gold particle-coated DNA vaccine.
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Introduction
Ideal cancer treatment should be able to eradicate systemic tumors at multiple sites of the body while having the specificity to discriminate between neoplastic and non-neoplastic cells. In this regard, antigen-specific cancer immunotherapy represents an attractive approach. The activation of antigen-specific T cell-mediated immune responses allows for the killing of tumors associated with a specific antigen and has become an important strategy for cancer immunotherapy.1, 2
The DNA vaccine has emerged as a novel method and an attractive strategy for the generation of antigen-specific cancer vaccines and immunotherapy. It is a new and powerful approach to generate immunologic responses against various diseases.3, 4, 5, 6 A variety of human clinical trials and animal models using DNA vaccines have been carried out for various infectious diseases,7, 8 therapies against cancer9, 10 and therapies against autoimmune diseases and allergies.11, 12 In addition, they have also become a widely used laboratory tool for a variety of applications ranging from proteomics to understand antigen presentation and cross-priming. The use of DNA vaccine technology precludes the need for handling hazardous viral pathogens, as only the DNA encoding antigens are incorporated into the vaccine.13 DNA vaccine technology also eliminates the need for biocontainment and the risk of exposure to live viral agents. Several modalities have been employed to deliver DNA vaccines, including intramuscular injection using conventional needle and syringe,3 electroporation,14 intradermally by the needle-free biojector15 and epidermally through a gene gun.16, 17
However, one of the concerns regarding DNA vaccines is their limited potency. This is characterized by two major properties: the low level of the antigen expression and the long-lasting expression. These two factors are believed to be responsible for the vaccination effect, which leads to a continuous stimulation of the immune system and training of memory cells.18 Our earlier work has shown that a chimeric DNA vaccine coated with gold particles using the gene gun approach to rout the human papillomavirus (HPV) type 16 E7 model antigen results in enhanced E7-specific CD8+ T cell-mediated immune responses and antitumor effects through different strategies, including targeting antigens by fusing molecules to enhance antigen processing,19 directing antigens to APCs by fusion to ligands for APC receptors,20 or to a pathogen sequence, such as domains of exotoxin in Pseudomonas,10, 21 intercellular spread22 and prolonging dendritic cells (DCs) survival through antiapoptotic molecules.9
Safety issues and complicated preparation are two important concerns for the clinical application of gene gun-delivered gold particle-coated DNA vaccines. So, we utilized a new, low-pressured gene gun to evaluate whether a noncarrier naked DNA vaccine can be delivered efficiently by this method and to evaluate the possibility of using noncarrier naked DNA vaccines without gold particle coating in the development of cancer vaccines and immunotherapy. We first showed that noncarrier naked DNA could be delivered into the cells of the intradermal layer. The noncarrier naked chimeric CRT/E7 DNA vaccine could also enhance antigen-specific T cell immunity and antibody response, and generate as potent antitumor effects as the gold particle-coated chimeric CRT/E7 DNA vaccine did. In addition, the burning effect on bombarded skin was less on noncarrier naked DNA vaccinated mice than that on gold particle-coated DNA vaccinated mice. We concluded that noncarrier naked DNA vaccines can be delivered by a low-pressured gene gun to generate similar potent antigen-specific immunities and antitumor effects, but with fewer side effects compared with gold particle-coated DNA vaccines. This can help improve the utility of naked DNA vaccines and promote human clinical trials of naked DNA vaccines in cancer vaccines and immunotherapy in the future.
Results
Fluorescence examination on skins of noncarrier naked pEGFP-N2 DNA-vaccinated mice
We first evaluated whether noncarrier naked DNA could be delivered by a low-pressure gene gun to the intradermal cells that express the interested gene. Very few intradermal cells expressed green fluorescence protein (GFP) after being immunized with noncarrier naked pEGFP-N2 DNA vaccine in various solution media after 6 hours (Figures 1a–d). However, significantly higher numbers of intradermal cells expressed GFP protein after immunization with noncarrier naked pEGFP-N2 DNA vaccine in various solution media after 24 h (Figures 1e–h). The areas of distribution for GFP-expressed cells did not reveal a difference between different volumes or formulations (data not shown). The GFP-expressing cells showed a cluster phenomenon in noncarrier naked DNA regardless of the solution medium (Figures 1f–h). However, the epidermal growth factor (EGF)-expressing cells showed a scattered phenomenon in the gold particle-coated DNA group (Figure 1e).
Fluorescence studies on skins of gold particle-coated or noncarrier naked pcDNA3-green fluorescence protein (GFP) DNA vaccinated mice. (a) Gold particle-coated DNA 6 h after immunization. (b) Noncarrier naked DNA in distilled H2O (ddH2O) solution 6 h after immunization. (c) Noncarrier naked DNA in phosphate-buffered saline (PBS) 6 h after immunization. (d) Noncarrier naked DNA in Tris-EDTA (TE) solution 6 h after immunization (note: only a few cells in the intradermis expressed the GFP protein). (e) Gold particle-coated DNA 1 day after immunization. (f) Noncarrier naked DNA in ddH2O solution 1 day after immunization. (g) Noncarrier naked DNA in PBS solution 1 day after immunization. (h) Noncarrier naked DNA in TE solution 1 day after immunization (note: most of the cells in the intradermis expressed the GFP protein).
Our results indicated that noncarrier naked DNA vaccine dissolved in various solutions, without gold particle coating, and could be delivered into the intradermis of mice to express the interested gene.
Vaccination with noncarrier naked CRT/E7 DNA four times generated the most optimal immunologic profiles and tumor preventive effects
We then determined the optimal number of vaccinations with noncarrier naked DNA to generate the most potent immune responses and antitumor effects. As shown in Figure 2a, the number of E7-specific CD8+ T-cell precursors increased when the number of noncarrier naked CRT/E7 DNA vaccinations increased (10.5±1.4 for naïve, 47.0±7.0 for twice, 103.0±9.9 for thrice, 157.5±8.5 for four times, P<0.001, one-way analysis of variance (ANOVA)). In addition, the titers of anti-E7 antibodies also correlated with the number of noncarrier naked CRT/E7 DNA vaccinations (Figure 2b; in 1:100 dilution, 0.447±0.017 for naïve, 0.730±0.035 for twice, 1.550±0.047 for thrice and 1.688±0.094 for four times, P<0.01, one-way ANOVA).
Immunologic profiles and in vivo tumor preventive effects of noncarrier naked CRT/E7 DNA vaccine with various times of vaccination. (a) Numbers of E7-specific CD8+ T-cell precursors (note: the frequencies of E7-specific CD8+ T-cell precursors correlated with the frequency of noncarrier naked CRT/E7 DNA vaccinations). (b) Titers of anti-E7 antibodies (note: the titers of anti-E7 antibodies increased when the number of noncarrier naked CRT/E7 DNA increased). (c) In vivo tumor preventive experiments (note: only 40 and 80% of mice receiving twice and thrice, respectively, noncarrier naked CRT/E7 DNA vaccine remained tumor-free for 60 days onward when challenged with TC-1 tumor cells). However, 100% of the mice that received noncarrier naked CRT/E7 DNA vaccine four times remained tumor-free 60 days after TC-1 tumor challenge.
For in vivo tumor protection experiments, all of the mice vaccinated four times with noncarrier naked CRT/E7 DNA vaccine were tumor-free after 60 days of TC-1 challenge. Only 40 and 80% of mice, which were vaccinated twice and thrice, respectively, remained tumor-free for 60 days onward when challenged with TC-1 tumor cells (Figure 2c).
Our results indicated that the number of noncarrier naked DNA vaccinations influenced the immune responses and tumor protective effects.
Noncarrier naked and gold particle-coated CRT/E7 DNA vaccines generate similar E7-specific immunity and antitumor effects
We further evaluated whether noncarrier naked DNA vaccine generates similar antigen-specific immunities and antitumor effects as gold particle-coated DNA vaccine. Mice vaccinated with noncarrier naked or gold particle-coated CRT/E7 DNA generated higher frequencies of E7-specific interferon (IFN)-γ-secreting CD8+ T-cell precursors when compared with mice vaccinated with the noncarrier naked or gold particle-coated wild-type E7 DNA (12.0±2.8 for noncarrier naked wild-type E7 group, 162.0±10.5 for noncarrier naked CRT/E7 group, 14.0±2.8 for gold particle-coated wild-type E7 group, 226.0±10.0 for gold particle-coated CRT/E7, P<0.05, one-way ANOVA; Figure 3a). The numbers of E7-specific CD8+ T-cell precursors in noncarrier naked CRT/E7 DNA vaccinated mice were fewer than those in the gold particle-coated CRT/E7 DNA vaccinated group (P<0.05, one-way ANOVA).
Immunologic profiles and tumor protection effects of noncarrier naked and gold particle-coated DNA vaccines. (a) Numbers of E7-specific interferon (IFN)-γ-secreting CD8+ T-cell precursors/3.5 × 105 splenocytes of various DNA vaccinated mice: 1, naive; 2, naked no insert; 3, naked E7; 4, naked CRT; 5, naked CRT/E7; 6, gold particle-coated CRT/E7 (note: mice vaccinated with noncarrier naked or gold particle-coated CRT/E7 DNA generated higher frequencies of E7-specific IFN-γ-secreting CD8+ T-cell precursors when compared with the other groups of mice). (b) Enzyme-linked immunosorbent assay (ELISA) showed E7-specific antibodies in mice vaccinated with various DNA vaccines (note: the titers of anti-E7 antibodies generated by the noncarrier naked or gold particle-coated CRT/E7 DNA showed significantly higher titers of anti-E7 antibodies compared with the other groups). (c) In vivo tumor preventive experiments for tumor-free. (d) In vivo tumor preventive experiments for survival of animals (note: both groups of mice that received noncarrier naked and gold particle-coated CRT/E7 DNA remained tumor-free 60 days, and were still alive 90 days after TC-1 challenge.
As shown in Figure 3b, mice vaccinated with noncarrier naked or gold particle-coated CRT/E7 DNA also showed significantly higher titers of anti-E7 antibodies compared with the wild-type E7 groups (for 1:100 dilution, naive 0.507±0.019; noncarrier naked wild-type E7 0.496±0.021; noncarrier naked CRT/E7 DNA 1.510±0.149; gold particle-coated wild-type E7 0.511±0.015; gold particle-coated CRT/E7 DNA 1.742±0.223; P<0.01, one-way ANOVA). However, there was no significant difference in the titers of E7 antibodies between the noncarrier naked and gold particle-coated CRT/E7 DNA groups (P>0.05, one-way ANOVA; Figure 3b).
We further carried out in vivo tumor protection experiments to compare the antitumor effects between noncarrier naked and gold particle-coated CRT/E7 DNA vaccines. As shown in Figures 3c and d, all of the mice, which received noncarrier naked or gold particle-coated CRT/E7 DNA vaccines remained tumor-free 60 days and were still alive 90 days after TC-1 challenge. In contrast, the unvaccinated, noncarrier naked no insert DNA, noncarrier naked E7 DNA, noncarrier naked CRT DNA and noncarrier naked E7 mixed with CRT DNA groups developed tumors within 20 days and died within 60 days of tumor challenge.
Our results indicated that mice vaccinated with noncarrier naked CRT/E7 DNA vaccine could enhance similarly potent E7-specific immunities and protective antitumor effects from E7-expressing TC-1 tumor challenge as gold particle-coated CRT/E7 DNA vaccine.
Noncarrier naked CRT/E7 DNA in various media generated E7-specific T-cell immunity and antitumor effects
We then evaluated whether the solution media influenced immune responses and antitumor effects. The representative figures of flow cytometric analysis of E7-specific IFN-γ-secreting CD8+ T-cell precursors are shown in Figure 4a. The numbers of E7-specific CD8+ T-cell precursors of noncarrier naked CRT/E7 DNA vaccinated mice, regardless of solution, were higher than those in naïve mice (11.5±2.1; P<0.05, one-way ANOVA). However, mice vaccinated with noncarrier naked CRT/E7 DNA in distilled H2O (ddH2O) (157.5±8.5) or Tris-EDTA (TE) (150.0±5.0) generated higher numbers of E7-specific CD8+ T-cell precursors compared to those immunized with noncarrier naked CRT/E7 DNA in phosphate-buffered saline (PBS; 94.0±4.0; P<0.05, one-way ANOVA; Figure 4b). As shown in Figure 4c, the titers of anti-E7 antibodies in mice vaccinated with noncarrier naked CRT/E7 DNA vaccine, regardless in which medium, were significantly higher than those in naive mice. However, the titers of anti-E7 antibodies were not different in mice vaccinated with noncarrier naked CRT/E7 DNA vaccine in different media (P>0.05, one-way ANOVA; Figure 4c).
Immunologic profiles and in vivo tumor preventive effects of noncarrier naked CRT/E7 DNA vaccine dissolved in various media and bombarded at various pressures. (a) Representative figures of flow cytometry analysis of E7-specific CD8+ T-cell precursors. (b) Frequencies of E7-specific CD8+ T-cell precursors of noncarrier naked CRT/E7 DNA vaccine in various solutions (note: the numbers of E7-specific CD8+ T-cell precursors for noncarrier naked CRT/E7 DNA vaccinated mice in distilled H2O (ddH2O) or Tris-EDTA (TE) solution were higher than those in phosphate-buffered saline (PBS) solution (P<0.05, one-way analysis of variance (ANOVA)). (c) Titers of anti-E7 antibodies in mice vaccinated with noncarrier naked CRT/E7 DNA in various media (note: the titers of anti-E7 antibodies were not significantly different between the noncarrier naked CRT/E7 DNA groups, regardless of which solution used (P>0.05, one-way ANOVA). (d) In vivo tumor preventive experiments in mice vaccinated with noncarrier naked CRT/E7 DNA vaccine in various solutions (note: mice vaccinated with noncarrier naked CRT/E7 DNA vaccine in ddH2O or TE were 100% tumor-free after 60 days of TC-1 challenge). However, only 60% of mice vaccinated with noncarrier naked CT/E7 DNA vaccine in PBS were tumor-free after 60 days of TC-1 challenge. (e) In vivo tumor preventive experiments in mice that received noncarrier naked CRT/E7 DNA at various pressures (note: mice vaccinated at 50 or 60 psi were 100% tumor-free after 60 days of TC-1 challenge). However, only 40 and 60% of mice were tumor-free after being vaccinated at 30 and 40 psi, respectively.
We further evaluated whether the immunity of noncarrier naked CRT/E7 DNA vaccine in various solution media could be translated to the antitumor effects. As shown in Figure 4d, 100% of the mice were tumor-free 60 days after TC-1 challenge when vaccinated with noncarrier naked CRT/E7 DNA vaccine in ddH2O or TE buffer. However, only 60% of the mice were tumor-free after 60 days of TC-1 challenge when given noncarrier naked CRT/E7 DNA vaccine in PBS buffer.
Our results indicated that DNA vaccine in different solution media could generate antigen-specific immunity and potent antitumor effects through the delivery of a low-pressured gene gun, and that ddH2O or TE solution could be more suitable media for the noncarrier naked DNA vaccine.
Noncarrier naked CRT/E7 DNA vaccinated with at least 50 psi was optimal for tumor protection against TC-1 E7-expressing tumor cells
To determine whether the delivery pressure in vaccination influenced the effect of noncarrier naked DNA vaccine, mice were vaccinated at various psi and challenged with TC-1 tumor cells to evaluate the antitumor effects. As shown in Figure 4e, only 40–60% of mice were tumor-free after 60 days of TC-1 challenge when given noncarrier naked DNA vaccine at 30 or 40 psi. However, when vaccinated at 50 or 60 psi, all of the mice were tumor-free 60 days after TC-1 challenge.
Our results indicated that the bombardment pressure of the gene gun influenced the potency of the noncarrier naked DNA vaccine.
The bombardment pressure influences the expression of the interested gene in vivo
The protein expressions of the interested gene at different bombardment pressures were further evaluated. The protein expression of luciferase was significantly lower in the dermis of noncarrier naked luciferase DNA immunized mice with 30 psi bombardment pressure compared to those with higher bombardment pressures (P<0.05, one-way ANOVA; Figure 4f).
Our results indicated that mice receiving noncarrier naked DNA vaccine with higher bombardment pressures can express higher amounts of protein of the interested gene.
Treatment with noncarrier naked CRT/E7 DNA vaccine led to a significant reduction of pulmonary tumor nodules
We further assessed the therapeutic potential of noncarrier naked CRT/E7 DNA vaccine using a lung hematogeneous spread model. The representative pulmonary tumor nodules in various vaccinated groups are shown in Figure 5a. As shown in Figure 5b, mice treated four times with noncarrier naked or gold particle-coated CRT/E7 DNA showed significantly less pulmonary tumor nodules than the other groups (naïve 92.4±7.4, noncarrier no insert 89.2±5.4, noncarrier naked wild-type E7 90.4±6.1, noncarrier naked CRT/E7 1.6±1.1 and gold particle-coated CRT/E7 0.8±0.8; P<0.001, one-way ANOVA). In addition, mice that received more vaccinations of noncarrier naked CRT/E7 DNA had lower numbers of pulmonary tumor nodules (twice 32.4±4.3, thrice 15.2±3.6, 4 times 1.6±1.1; P<0.01, one-way ANOVA). However, the number of pulmonary tumor nodules was not different between mice immunized four times with noncarrier naked and those four times with gold particle-coated CRT/E7 DNA vaccine (1.6±1.1 vs 0.8±0.8; P>0.05, one-way ANOVA).
In vivo tumor treatment experiments in mice that received a high therapeutic dose of noncarrier naked or gold particle-coated DNA vaccine. (a) Representative figures of pulmonary tumor nodules in each group: 1, naïve; 2, no insert; 3, E7; 4, naked CRT/E7 twice; 5, naked CRT/E7 thrice; 6, naked CRT/E7 four times; 7, gold particle-coated CRT/E7. (b) Mean pulmonary tumor nodules in mice vaccinated with different frequencies of noncarrier naked CTGFCRT/E7 DNA vaccine and gold particle-coated CRT/E7 DNA vaccines (note: mice that received the noncarrier naked CRT/E7 DNA vaccine had less pulmonary tumor nodules than those that received the noncarrier naked wild-type E7 DNA vaccine). Mice that were treated with noncarrier naked CRT/E7 DNA four times showed significantly less pulmonary tumor nodules than those that were treated twice or thrice. The number of pulmonary tumor nodules was not different between mice immunized with noncarrier naked and gold particle-coated CRT/E7 DNA vaccine four times. (c) Mean pulmonary tumor nodules in mice vaccinated with noncarrier naked CRT/E7 DNA in various media (note: the mean pulmonary tumor nodules of mice vaccinated with noncarrier naked CRT/E7 DNA in distilled H2O (ddH2O) or Tris-EDTA (TE) solution was significantly lower than those in mice treated with noncarrier naked DNA vaccine in phosphate-buffered saline (PBS).
Our results indicated that mice vaccinated more times with noncarrier naked DNA vaccine generated more potent therapeutic effects. In addition, mice vaccinated with noncarrier naked or gold particle-coated DNA vaccine generated similarly excellent therapeutic effects.
Noncarrier naked CRT/E7 DNA in various media generated different therapeutic effects
We then evaluated whether the solution media influenced the therapeutic effects of noncarrier naked CRT/E7 DNA vaccine. As shown in Figure 5c, mice treated with noncarrier naked CRT/E7 DNA in ddH2O (1.6±0.5) or TE solution (2.6±1.8) had less numbers of pulmonary tumor nodules compared to those in PBS solution (19.8±2.1; P<0.05, one-way ANOVA).
Our results indicated that different solution media influenced the therapeutic effects of noncarrier naked DNA vaccine.
CD11c-enriched cells from mice vaccinated with noncarrier naked CTGF/E7 DNA effectively enhanced the activation of an E7-specific CD8+ T-cell line
Several studies have shown that following intradermal immunization, DCs migrate to draining lymph nodes where they play a major role in priming and stimulating antigen-specific T cells.23, 24 Therefore, it is important to determine whether DCs express the reporter gene in draining lymph nodes when noncarrier naked DNA is administered. The draining lymph nodes of mice vaccinated with various DNA vaccines and CD11+ cells were first isolated as described in Materials and methods. The CD11c-enriched cells were then incubated with an E7-specific T-cell line to evaluate their ability to stimulate INF-γ secretion. The representative figures of flow cytometric analysis are shown in Figure 6a. As shown in Figure 6b, CD11c-enriched cells isolated from mice vaccinated with noncarrier naked and gold particle-coated CRT/E7 DNA vaccines were more effective in activating the E7-specific CD8+ T-cell line to secrete IFN-γ compared with noncarrier naked wild-type E7 DNA vaccine (70±15 for E7 group, 419±11 for naked CRT/E7 in ddH2O group, 405±12 for naked CRT/E7 in PBS group, 412±14 for naked CRT/E7 in TE group, 380±19 for gold particle-coated CRT/E7 group; P<0.001, one-way ANOVA). However, there was no difference in the numbers of IFN-γ-secreting CD8+ T cells between gold particle-coated and noncarrier naked CRT/E7 DNA vaccines in respective solution media (P>0.05, one-way ANOVA). These results are consistent with the notion that in vivo DNA-transfected DCs from mice vaccinated with CRT/E7 DNA can enhance the activation of E7-specific CD8+ T cells regardless of the noncarrier naked or gold particle-coated groups.
Activation of E7-specific CD8+ T cells by CD11c-enriched cells isolated from the inguinal lymph nodes of noncarrier naked or gold particle-coated DNA vaccinated mice. (a) Representative figures of flow cytometry analysis. (b) Numbers of interferon (IFN)-γ-secreting E7-specific CD8+ T cells stimulated by CD11c-enriched cells isolated from the inguinal lymph nodes of vaccinated mice (note: CD11c-enriched cells isolated from mice vaccinated with noncarrier naked and gold particle-coated CRT/E7 DNA vaccines were more effective in activating the E7-specific CD8+ T-cell line to secrete IFN-γ compared with noncarrier naked wild-type E7 DNA vaccine). However, there was no difference in the numbers of IFN-γ-secreting CD8+ T cells between gold particle-coated and noncarrier naked CRT/E7 DNA vaccines in respective solution media. The data collected from all of the above experiments are from one representative experiment of the two experiments that were carried.
Noncarrier naked DNA vaccine generated less burning effect on the skin of mice compared with gold particle-coated DNA vaccine
We finally evaluated whether the local skin reaction would be similar on mice bombarded with noncarrier naked and gold particle-coated chimeric DNA vaccines. As shown in Figures 7b, d, f, and h, no definite skin damage could be identified in mice vaccinated with noncarrier naked DNA. However, local skin damage was significant from 1 day after the gold particle-coated DNA vaccination (Figure 7e). The most severe skin damage was noted 3 days after vaccination (Figure 7g), and it took 7 days for the skin to heal (Figure 7i).
Burning effects on the skin of noncarrier naked and gold particle-coated DNA vaccinated mice. Mice were first given noncarrier naked CRT/E7 chimeric DNA or gold particle-coated CRT/E7 DNA at 50 psi as described in the Materials and methods section. (a) Shaved skin before vaccination. (b) Mice that received noncarrier naked DNA vaccine immediately. (c) Mice that received gold particle-coated DNA vaccine immediately. (d) Mice that received noncarrier naked DNA vaccine 24 h later. (e) Mice that received gold particle-coated CRT/E7 DNA vaccine 24 h later. (f) Mice that received noncarrier naked DNA vaccine after 3 days. (g) Mice that received gold particle-coated DNA vaccine after 3 days. (h) Mice that received noncarrier naked CRT/E7 DNA vaccine after 7 days. (i) Mice that received gold particle-coated CRT/E7 DNA vaccine after 7 days (note: the skin damage was noted from 24 h after being given the gold particle-coated DNA vaccine and the damage continued until 7 days postimmunization. There was no visible skin damage for mice that received noncarrier naked DNA immunization.
Our observation indicated that noncarrier naked DNA vaccination resulted in significantly less local skin damage than gold particle-coated DNA vaccination.
Discussion
DNA vaccination is a new and powerful approach to generate immunologic responses. Only a small quantity of DNA vaccine is required to enhance in vivo immune responses. Gregersen25 showed that intradermal gene gun immunization needs smaller quantities of DNA compared with intramuscular injections,25 which always requires large amounts of DNA (100 μg per mouse) to elicit the immune response. In contrast, the microparticulated bombardment system can induce an immune response using very small amounts of DNA (1 μg per mouse).26 In addition, gene gun immunization has a 10- to 100-fold greater expression of the DNA-encoded protein than intramuscular vaccinations.27, 28
Gold particle-coated DNA vaccines with a gene gun delivery system are currently widely used in immunopreventive and immunotherapeutic applications. Traditionally, DNA vaccines need to be coated with gold particles to be heavy enough to be bombarded into the intradermis and deliver the antigens of interest to the intradermal Langerhan’s cells (antigen-presenting cells) to induce potent immunologic responses and antitumor effects. High efficiency and stability are two key points in these DNA vaccines coated with gold particles for in vivo use. Particle-mediated gene gun technology uses compressed helium to propel micrometer-sized colloidal gold particles coated with plasmid through the stratum corneum, where the particles lodge in the epidermal and dermal layers of the skin.29 The gene gun technique has been used to immunize various animals for tumor prevention and regression, including prostate cancer, melanomas and cervical cancer.25, 30 The additional advantage of gene gun immunization is the application to a broad variety of cells, including Langerhan’s cells, DCs and keratinocytes, which are transfected.31 The DNA vaccine can then provide protein expression in the transfected cells, and the transfected Langerhan’s cells and dermal DCs can migrate to local lymph nodes where presentation of antigens to T cells can occur, and thus start a variety of immunologic responses.29, 32
Inconvenience in preparation and safety issues are concerns for the clinical applications of gene gun-delivered, gold particle-coated DNA vaccines. The conventional gene gun delivery system can only deliver dry DNA bullets instead of wet DNA bullets because of inherent limitations. In contrast, the noncarrier naked DNA vaccine can be delivered in the form of wet DNA bullets. Moreover, the preparation of gold particle-coated DNA vaccines is hardly aseptic. However, the preparation of noncarrier naked DNA vaccines can be relatively aseptic and simpler. As such, safety concerns of gold particle-coated DNA vaccine is a major consideration in human clinical trials. Another disadvantage of gold particle-coated DNA vaccine is that non-biodegradable gold or tungsten may skew the immune response or cause adverse side effects when they accumulate.33 Finally, gold particle-coated DNA vaccination always induces burning effects on bombarded skin, as shown presenting this and in earlier studies.17, 34, 35 The novel technology of noncarrier naked DNA vaccination could resolve these above-mentioned concerns.
Noncarrier naked DNA vaccine can be as effective as gold particle-coated DNA vaccine to reach intradermal layer, to deliver the antigens of interest to the intradermal Langerhan’s cells and to enhance the subsequent immune responses. Our earlier results have shown that gold particle-coated CRT/E7 DNA vaccine could generate more potent immune responses and antitumor effects than gold particle-coated wild-type E7 DNA vaccine.34 We observed that noncarrier naked CRT/E7 DNA vaccine generates less numbers of E7-specific CD8+ T-cell precursors than gold particle-coated CRT/E7 DNA vaccine, although 100% tumor protection effects were seen in both DNA formats (Figures 2a and 3c). We hypothesize that gold particle-coated DNA vaccination may induce non-specific immune responses to enhance the antigen-specific immune responses by the local burning effect on the bombarded skin, as shown in our earlier studies.19, 51 Another possible mechanism is that the gold beads alone can upregulate the antigen-presenting cells,36 and these upregulated DCs can induce more potent antigen-specific immune responses when encountering specific antigens.
Different solution media can influence the antigen-specific immunity and antitumor effects of noncarrier naked DNA vaccine. Our results indicated that noncarrier naked CRT/E7 DNA in ddH2O or TE buffer can generate better E7-specific immune responses and antitumor effects than in PBS buffer (Figures 4b and d). Hartikka et al.37 observed that the concentration, osmolarity and pH of sodium phosphate can influence plasmid DNA expression in vivo. The optimum sodium phosphate concentration was 150 mM and the luciferase expression was 4.3-fold compared with saline. The luciferase expression decreased significantly, when solved in sodium phosphate solution less than 40 mM, compared to that of saline.37 The sodium concentration of PBS buffer was only 6.7 mM in this study. This might be the reason why the PBS group did not generate as potent antigen-specific immune responses and antitumor effects as the other two groups in this study. It is essential to test and identify the optimal buffer and its concentration for noncarrier naked DNA vaccine.
Dermal immunization methods target the epidermis, the dermis, or both, and include chemical modification,38, 39 transepidermal immunization,40, 41 gene gun technology,31 electroporation42, 43 and intradermal injections.42 Recently, intradermal administration with the RNA interference technique has also been reported.44 The benefit of intradermal administration by the gene gun technique is that it is less harmful and injurious to the host. Hirao et al.42 observed higher cellular and humoral responses to an HIV DNA vaccine with electroporation compared with intradermal route alone. Electroporation may have importance as an immunization approach in larger animal models. More studies are needed to identify the optimal immunization approach in different animal models.
Multiple vaccinations are always needed for both noncarrier naked and gold particle-coated DNA vaccines delivered by a gene gun. Gold particle-coated, and especially, noncarrier naked DNA vaccine needs boosters to enhance immune responses and antitumor effects. One of the benefits of the dermal immunization technique is that it is needleless. However, the disadvantage is the requirement of frequent and multiple sites per immunization to elicit immune responses. Doria-Rose et al.45 reported that a prime-boost strategy seems necessary for DNA vaccines. We also observed that more shots of noncarrier naked DNA vaccine were needed to generate comparable immune responses and antitumor effects compared with gold particle-coated DNA vaccine. Gregersen reported that frequent or multiple sites of application may be safe and acceptable from a scientific point of view, although they may not necessarily be tolerated and widely accepted by patients.25 Peng et al. recently reported that cluster intradermal antigen-specific gold particle-coated DNA vaccination is capable of rapidly inducing antigen-specific CD8+ T-cell immune responses leading to therapeutic anti-tumor effects.46 The noncarrier naked DNA vaccine can also use a similar protocol to induce potent anti-tumor immune responses in a shorter interval.
Noncarrier naked DNA vaccine induces less tissue injury as compared to gold particle-coated DNA vaccine, where the gold particles can cause slight necrosis and mild to moderate tissue damage. Pilling et al. reported that gold particle-coated DNA bombardment with the gene gun system induced local skin reactions two days post-dosing in mini-pigs.47 By 28 days, the skin lesions had regressed apart from a low grade peri-vascular mononuclear cell infiltration in the upper dermis, which persisted up to 141 days, together with a small number of phagocytosed gold particles.47 We also observed that gold particle-coated DNA vaccine bombardment by a gene gun has definite local skin damage as compared to noncarrier naked DNA vaccine (Figure 6). In addition, the most severe local skin damage of the mice was seen three days after bombardment with gold particle-coated DNA vaccine. The bombardment pressure might be a factor in generating skin damage. DNA vaccines are always administered in 1–8 vaccinations on non-overlapping sites on shaved epidermis using 150–1200 psi with a majority of investigators favoring around 400 psi of helium pressure.48 We bombarded the mice with a lower pressure of 50 psi of helium pressure for both gold particle-coated and noncarrier naked DNA vaccines in multiple sites on the shaved abdomen in this study. Our observations imply that noncarrier naked DNA vaccine could be effectively delivered by an intradermal gene gun at lower helium pressure so that damage to local skin can be minimized.
E7-specific antibody titers were significantly enhanced by noncarrier naked and gold particle-coated CRT/E7 DNA vaccines, although the mechanism for enhancement of antibody responses by CRT/E7 DNA vaccine is not clear. The titers of anti-E7 antibodies were similar between noncarrier naked and gold particle-coated DNA vaccines in this study. We also observed that the CRT/E7 proteins could be detected in the sera,34 and the levels of CRT/E7 proteins were similar between noncarrier naked and gold particle-coated DNA vaccinated groups (data not shown). We hypothesize that the similar amounts of secreted CRT/E7 proteins, regardless of whether generated by noncarrier naked or gold particle-coated CRT/E7 DNA vaccine, might enhance the humoral-mediated immune response to generate similar anti-E7 antibodies. Even though antobody-mediated responses have not been shown to play an important role in controlling HPV-associated malignancies, antigen-specific antibodies are significant in other tumor models such as the breast cancer model with the HER-2/neu antigen. Lin et al. reported that noncarrier naked neu DNA vaccine can generate anti-neu antibodies and anti-tumor effects as much as gold particle-coated neu DNA vaccine,49 and the titers of anti-neu antibodies correlated with the doses of noncarrier naked DNA vaccination.49 The chimeric CRT/E7 DNA vaccine, regardless of whether in the noncarrier naked format, can significantly enhance higher titers of anti-E7 antibodies. This novel strategy can be utilized to generate the other oncogene-specific antibodies for the treatment of oncogene overexpressing tumor model.
In summary, our study showed that noncarrier naked chimeric CRT/E7 DNA vaccine, without gold particle coating, bombarded under low pressure by a gene-gun delivery system, can be delivered into the intradermis of mice to express the interested gene and enhance similar E7-specific immunities and antitumor effects compared with the gold particle-coated CRT/E7 DNA vaccine.
Materials and methods
Plasmid DNA constructs and preparation
The generation of pcDNA3 with no insert as well as pcDNA3-E7, pcDNA3-CRT, pcDNA3-CRT/E7 and pcDNA3-CRT/E7/GFP has been described earlier.34 pEGFP-N2 was purchased from Clontech Laboratories (Palo Alto, CA, USA).
Mice
Six- to eight-week-old female C57BL/6J mice were bred in, and purchased from the animal facility of the National Taiwan University Hospital (Taipei, Taiwan). All animal procedures were carried out according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.
DNA vaccination
For vaccination of noncarrier naked DNA or gold particle-coated DNA, a low pressure-accelerated gene gun (BioWare Technologies Co. Ltd, Taipei, Taiwan) was used as described previously.9, 49 Mice were immunized with respective DNA vaccine four times at 1 week intervals. Mice (five per group) were immunized with 2 μg of noncarrier naked or gold particle-coated DNA vaccine each time.
To test the influence of dissolved media in DNA vaccination, noncarrier chimeric pcDNA3-CRT/E7 or pEGFP-N2 DNA was dissolved in PBS (pH8.0), TE buffer (pH 7.0) or ddH2O for vaccination. To evaluate the influence of the bombardment pressure of the gene gun, mice were immunized with noncarrier DNA vaccine at various pressures.
Immunofluorescence examination of the skin of noncarrier naked DNA vaccinated mice
The mice's (five per group) abdomens were shaved for the DNA vaccination (pEGFP-N2 in dd-H2O, PBS or TE buffer, respectively) through the gene gun at 50 psi as described earlier. Six or 24 h after, the mice were killed so that the abdominal skin could be observed under fluorescence microscope (BX60, Olympus Corp., Tokyo, Japan).
The detection of luciferase assay within the skin of mice vaccinated with various bombardment pressures
Mice were prepared and bombarded with the pCMV-Luciferase DNA vaccine at various bombardment pressures of the gene gun as described earlier. One day after DNA immunization, the mice were killed and the bombarded area of the abdominal skin was taken off and then homogenized with lysis buffer (Promega, Madison, WI, USA) into sample lysates. The lysates were centrifuged and the supernatant was collected for further analysis. The supernatants were then added to a 96-well plate, and an equal volume of Bright-Glo (Promega) luciferase assay reagent was added. The results were counted with a luminometer (TopCount NXT, PerkinElmer, Meriden, CT, USA). The known concentrations of recombinant luciferase (Promega) were used as standards to calculate the luciferase content in each sample. The contents of total protein in the samples were also assayed by using a BCA assay kit (Pierce, Rockford, IL, USA). Finally, the luciferase content in each sample was normalized by the total protein content.
Intracellular cytokine staining and flow cytometry analysis
Mice (five per group) were immunized with 2 μg of various noncarrier naked or gold particle-coated DNA vaccines, followed by a booster 1 week later, four times. Splenocytes were harvested 1 week later and incubated with either 1 μg ml−1 of E7 peptide (aa 49–57) overnight.50 Cell surface marker staining of CD8 and intracellular cytokine staining for IFN-γ, as well as flow cytometry analysis, were carried out as described previously.9
Enzyme-linked immunosorbent assay (ELISA) for anti-E7 antibody
Mice (five per group) were immunized with 2 μg of the various DNA vaccines and a booster with the same regimen 1 week later. Sera were prepared from mice on day 14 after immunization. For the detection of HPV 16 E7-specific antibodies in sera, direct ELISA was used as described previously.51, 52 Briefly, a 96-microwell plate was coated with 100 μl of bacteria-derived HPV-16 E7 proteins (0.5 μg ml−1) and incubated at 4 °C overnight. The wells were then blocked with PBS containing 20% fetal bovine serum. Sera were serially diluted in PBS, added to the ELISA wells and incubated at 37 °C for 2 h. After washing with PBS containing 0.05% Tween 20, the plate was incubated with a 1:2000 dilution of a peroxidase-conjugated rabbit antimouse immunoglobulin antibody (Zymed, San Francisco, CA, USA) at room temperature for 1 h. The plate was washed, developed with 1-step Turbo TMB-ELISA (Pierce, Rockford, IL, USA) and stopped with 1 M H2SO4. The ELISA plate was read with a standard ELISA reader at 450 nm.
TC-1 tumor cell line
The generation of TC-1 tumor cell line has been described previously.53 Briefly, HPV-16 E6, E7 and Ras were used to transform primary C57BL/6 mice lung epithelial cells. This tumorigenic cell line was named TC-1. The TC-1 cells were grown in RPMI-1640, supplemented with 10% (vol/vol) fetal bovine serum, 50 U ml−1 penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM non-essential amino acids, and 0.4 mg ml−1 G418 at 37 °C with 5% CO2. On the day of tumor challenge, tumor cells were harvested by trypsinization, washed twice with 1 × Hank’s buffered salt solution, and finally resuspended in 1 × Hank’s buffered salt solution to the designated concentration for injection.
In vivo tumor protection experiments
For the tumor protection experiments, mice (five per group) were immunized with 2 μg of various noncarrier naked or gold particle-coated DNA vaccines and boosted 1 week later for a total of four times with a gene gun. One week after the last vaccination, the mice were subcutaneously challenged with 5 × 104 cells per mouse of TC-1 tumor cells in the right leg.53 The mice were monitored for evidence of tumor growth by palpation and inspection twice a week until they were killed on day 60.
In vivo tumor treatment experiments
For in vivo tumor treatment experiments, the mice were injected with 5 × 104 cells per mouse TC-1 tumor cells through the tail vein as described previously.9 Two days after tumor challenge, the mice were given 16 μg per mouse of various noncarrier naked or gold particle-coated DNA vaccines, followed by a booster with the same regimen every 7 days for 4 weeks (a total of two 64 μg DNA). Mice that were not vaccinated were used as a negative control. The mice were killed and the lungs were explanted on day 28. Pulmonary tumor nodules in each mouse were evaluated and counted by experimenters blinded to the sample identity.
Preparation of CD11c+ cells in the inguinal lymph nodes from vaccinated mice
Mice (five per group) received multiple inoculations of non-overlapping intradermal administration with a gene gun on the abdominal region. The mice were vaccinated with 16 μg of noncarrier naked or gold particle-coated CRT/E7 DNA. Inguinal lymph nodes were harvested from vaccinated mice 3 days after vaccination with a gene gun. A single cell suspension from isolated inguinal lymph nodes was prepared as described previously.9 CD11c+ cells were enriched from lymph nodes using CD11c (N418) micro-beads (Miltenyi Biotec, Auburn, CA, USA). The purity of the CD11c+ cells were further characterized using phycoerythrin-conjugated anti-CD11c antibody (PharMingen, San Diego, CA, USA) and analyzed by flow cytometry analysis,9 and samples of more than 90% of the CD11c+ cells were utilized for the following experiments.
Activation of E7-specific CD8+ T-cell line by CD11c-enriched cells
CD11c-enriched cells (2 × 104) were incubated with 2 × 106 of the E7-specific CD8+ T-cell line for 16 h. The cells were then stained for both surface CD8 and intracellular IFN-γ and analyzed with flow cytometry analysis as described above.
Local skin burning effect on noncarrier naked and gold particle-coated DNA vaccines
To compare the local skin burning effect, mice were vaccinated with noncarrier naked or gold particle-coated CRT/E7 DNA vaccine as described earlier, and pictures of the shaved DNA-bombarded abdominal skin of the mice were taken before and after immunization at 1, 3, 5 or 7 days later.
Statistical analysis
The data expressed as mean±s.e.m. or mean±s.d. are representative of at least two different experiments. Data for intracellular cytokine staining with flow cytometry analysis and tumor treatment experiments were evaluated by ANOVA. Comparisons between individual data points were made using the Student's t-test. In the tumor protection experiment, the principal outcome of interest was the time to the development of a tumor. The event time distributions for different mice were compared by the Kaplan–Meier method, and by log-rank analysis.
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Acknowledgements
This study was supported by a grant from the New Century Health Care Promotion Foundation and in part by the Department of Medical Research of NTUH. The E7-specific CD8+ T-cell line and TC-1 tumor cell line were kindly provided by Dr TC Wu of Johns Hopkins Medical Institutes in Baltimore, MD, USA. The pCMV-Luciferase DNA was kindly provided by Dr MD Lai of National Cheng Kung University in Tainan, Taiwan.
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Chen, CA., Chang, MC., Sun, WZ. et al. Noncarrier naked antigen-specific DNA vaccine generates potent antigen-specific immunologic responses and antitumor effects. Gene Ther 16, 776–787 (2009). https://doi.org/10.1038/gt.2009.31
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DOI: https://doi.org/10.1038/gt.2009.31
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