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. 2009 May 5;17(8):1473–1481. doi: 10.1038/mt.2009.84

DNA/Amphiphilic Block Copolymer Nanospheres Promote Low-dose DNA Vaccination

Dorian McIlroy 1,*, Benoît Barteau 2,3,6, Jeannette Cany 4,5, Peggy Richard 2,3, Clothilde Gourden 6, Sophie Conchon 4,5, Bruno Pitard 2,3,6
PMCID: PMC2835232  PMID: 19417740

Abstract

Intramuscular (i.m.) DNA vaccination induces strong cellular immune responses in the mouse, but only at DNA doses that cannot be achieved in humans. Because antigen expression is weak after naked DNA injection, we screened five nonionic block copolymers of poly(ethyleneoxide)-poly(propyleneoxide) (PEO-PPO) for their ability to enhance DNA vaccination using a β-galactosidase (βGal) encoding plasmid, pCMV-βGal, as immunogen. At a high DNA dose, formulation with the tetrafunctional block copolymers 304 (molecular weight [MW] 1,650) and 704 (MW 5,500) and the triblock copolymer Lutrol (MW 8,600) increased βGal-specific interferon-γ enzyme-linked immunosorbent spot (ELISPOT) responses 2–2.5-fold. More importantly, 704 allowed significant reductions in the dose of antigen-encoding plasmid. A single injection of 2 µg pCMV-βGal with 704 gave humoral and ELISPOT responses equivalent to those obtained with 100 µg naked DNA and conferred protection in tumor vaccination models. However, 704 had no adjuvant properties for βGal protein, and immune responses were only elicited by low doses of pCMV-βGal formulated with 704 if noncoding carrier DNA was added to maintain total DNA dose at 20 µg. Overall, these results show that formulation with 704 and carrier DNA can reduce the dose of antigen-encoding plasmid by at least 50-fold.

Introduction

In mice, intramuscular (i.m.) injection of plasmid DNA elicits immune responses with a strong Th1 bias, including a robust cytotoxic T lymphocyte (CTL) response.1,2,3 This makes DNA vaccination an attractive technology for the production of new vaccines against viruses, intracellular parasites, and cancers because strong CTL responses cannot generally be obtained using recombinant proteins or other nonreplicating formulations, such as virus-like particles and inactivated viruses. However, the promising results obtained with DNA vaccination in mice have not been reproduced in humans. Numerous clinical trials have reported disappointing results, with most subjects developing weak or undetectable CTL responses despite multiple i.m. DNA vaccinations.4,5,6

In light of the limitations of naked DNA vaccines in clinical trials, many groups have proposed strategies to enhance the efficacy of DNA vaccination. A variety of different approaches have been adopted, falling into three broad categories. First, attempts have been made to increase the immunogenicity of the antigens themselves by expressing them as fusion proteins with the immunodominant domains of bacterial toxins7,8 to provide T-cell help, or heat-shock proteins to enhance cross-presentation by dendritic cells.9,10,11 Second, immunological adjuvants, including both chemical adjuvants,12,13 and plasmids coding for cytokines14,15 or chemokines16,17,18 have been tested for their ability to enhance DNA vaccination. Third, attempts have been made to enhance the expression of antigen for a given DNA dose, either by optimizing the codon usage of the expressed gene,19,20 or by the use of electroporation21 or chemical vectors22 to increase the uptake of plasmid DNA after inoculation.

Although few quantitative studies have been performed, it appears that the great majority of naked plasmid DNA injected i.m. is rapidly degraded23 and therefore cannot direct expression of antigen. This is a severe limitation, not only for standard DNA vaccination, but also for many of the strategies outlined above, as they depend on the expression of modified antigens or cytokines/chemokines encoded on plasmid DNA. Increasing the efficiency of DNA delivery into myotubes is therefore an important step in the development of effective i.m. DNA vaccination.

In the present work, we test different triblock and tetrafunctional block copolymers of poly(ethyleneoxide)-poly(propyleneoxide) (PEO-PPO) with various structures and molecular weights (MWs) for their ability to potentiate DNA vaccination in mice. The structure and biological properties of triblock copolymers have been extensively reviewed.24 They are unbranched linear polymers, with PEO groups attached to each extremity of a hydrophobic PPO core. In contrast, tetrafunctional copolymers have a structure consisting of four PEO-PPO blocks centered on an ethylenediamine moiety. Both triblock and tetrafunctional block copolymers have previously been shown to enhance gene delivery into many tissues,24,25,26 including skeletal muscle,27 and therefore seemed to be good candidates for nonviral vectors to improve i.m. DNA vaccination. We therefore sought to determine to what extent PEO-PPO block copolymers (i) enhance the immune response obtained with high-dose DNA vaccination and (ii) allow the dose of antigen-coding plasmid DNA to be reduced without affecting vaccine potency.

Results

Block copolymer 704 enhances IFNγ ELISPOT response to i.m. DNA vaccination

To test the potential of different block copolymers as adjuvants for DNA vaccination, we screened a panel of five different molecules previously shown to enhance the expression of reporter genes in vivo.25,26,27,28 Mice were injected on day 0 and day 21 with 70 µg pCMV-βGal [35 µg in each tibialis anterior (TA) muscle] either alone, or formulated with the following block copolymers: the triblock copolymers Lutrol (MW 8,600) and PE6400 (MW 2,900) and the tetrafunctional block copolymers 304 (MW 1,650), 704 (MW 5,500), and 904 (MW 6,600) (Figure 1a). Each polymer was used at the concentration that gave maximal enhancement of reporter gene expression as previously published25,26,27,28 or as shown in Supplementary Figure S1 for 704. Two mice per group were killed at day 7 after the first DNA injection to compare levels of β-galactosidase (βGal) expression in different groups (Figure 1b), whereas the humoral and cellular immune responses were followed in the remaining five mice.

Figure 1.

Figure 1

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Effect of different block copolymers on antigen expression and the immune response after high-dose DNA vaccination. (a) Schematic structure of tetrafunctional block copolymers 304 (y = 4, x = 4; MW 1,650), 704 (y = 13, x = 14; MW 5,500), and 904 (y = 15, x = 17; MW 6,600). (b–d) Groups of mice were injected intramuscularly with 70 µg pCMV-βGal (35 µg in each tibialis anterior (TA) muscle) either alone (Inline graphic) or formulated with 3% Lutrol (Lut, □), 0.05% PE6400 (PE, ⋄), 5% 304 (*), 0.25% 704 (▵), or 0.1% 904 (○). n = 7 for all groups except PE6400 n = 6. (b) Antigen expression. Two mice per group were killed at day 7 after the first DNA injection, and βGal expression in situ was monitored by coloration with X-Gal. One representative TA muscle of four is shown for each group. (c) Humoral response. Geometric mean titers (GMTs) are shown for each group. Arrows indicate DNA injection at day 0 and boost at day 21. For clarity, sera at day 0 with no detectable βGal-specific IgG are presented at a titer of 1/25. (d) Class I–restricted cellular response. Splenocytes were prepared at day 42, stimulated overnight with the ICPMYARV peptide, and the number of IFNγ spot-forming cells (SFCs) was determined. Each symbol represents the result from one mouse. Significant differences between groups are indicated. *P < 0.05; **P < 0.01 by analysis of variance and Tukey's least significant difference post hoc test.

Significant enhancement of βGal expression was observed with all formulations compared to naked DNA. Formulation with all five polymers gave an enhanced specific humoral response at day 14 (Figure 1c), although anti-βGal titers were equivalent in all groups including naked DNA after a booster vaccination at day 21. Class I–restricted cellular immune responses did not show the same relationship with antigen expression. DNA formulated with PE6400 or 904 gave equivalent enzyme-linked immunosorbent spot (ELISPOT) responses to naked DNA, whereas formulation with Lutrol, 304, and, in particular, 704 significantly enhanced the βGal-specific response (Figure 1d). Overall, DNA formulated with 704 gave the best combination of enhanced antigen expression, humoral and class I–restricted cellular immune responses, so subsequent experiments concentrated on this molecule.

Dose response of DNA vaccination with 704

Dose–response experiments using pCMV-βGal DNA formulated with 704 showed that maximal ELISPOT and humoral immune responses to βGal were maintained as DNA dose dropped from 100 to 20 µg but began to decline when DNA dose was further reduced to 4 µg (Supplementary Figure S2). However, it has been known for many years that noncoding plasmid DNA has immune adjuvant activity, principally due to the presence of hypomethylated immunostimulatory CpG motifs.29,30,31 Mice were therefore vaccinated on day 0 and day 21 with 20, 2, or 0.2 µg of pCMV-βGal formulated with 704, either alone or together with increasing quantities of the cloning vector pUC19 to maintain the total injected DNA dose at 20 µg. pUC19 was used as carrier DNA because it does not contain a eukaryotic promoter region and therefore should not interfere with the recruitment of transcription factors to the cytomegalovirus promoter of the co-injected pCMV-βGal plasmid. Without carrier DNA, immune responses were significantly reduced at 2 µg pCMV-βGal and undetectable at 0.2 µg pCMV-βGal (Figure 2a,c). In the presence of carrier DNA, however, maximal cellular and humoral immune responses were still observed using only 2 µg pCMV-βGal and even 0.2 µg pCMV-βGal induced a robust cellular response (Figure 2b,d).

Figure 2.

Figure 2

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Effects of carrier plasmid on DNA vaccination with 704. Groups of mice (n = 4) were injected intramuscularly with 20 µg (▴, ▵), 2 µg (Inline graphic, ⋄), 0.2 µg (•, ○), pCMV-βGal formulated with 0.25% 704 in the absence (a,c) or the presence (b,d) of pUC19 plasmid to maintain a total DNA dose of 20 µg per mouse. (a,b) Humoral response. Geometric mean titers are shown for each group ± standard deviation. Arrows indicate DNA injection at day 0 and boost at day 21. Figures on the right indicate the pCMV-βGal plasmid dose for each group. For clarity, sera with no detectable βGal-specific IgG are presented at a titer of 1/25. (c,d) Class I–restricted cellular response. Splenocytes were prepared at day 42, stimulated overnight with the βGal ICPMYARV peptide (▴, Inline graphic, •) or control peptide (▵, ⋄, ○), and the number of IFNγ SFCs was determined. Each symbol represents the result from one mouse. Data from the group injected with 20 µg pCMV-βGal are duplicated in panels a and c, and b and d. Significant differences between groups are indicated. *P < 0.05, **P < 0.01 by analysis of variance and Tukey's least significant difference post hoc test. SFC, spot-forming cell.

The pUC19 plasmid encodes the N-terminal α-fragment of βGal, and expression of this truncated protein, although unlikely, could theoretically contribute to the enhanced immunogenicity of DNA vaccination in the presence of pUC19. To exclude this possibility, subsequent experiments were carried out using the pQE30 plasmid, which does not contain βGal coding sequences. As shown in Figure 3, a single vaccination with 2 µg pCMV-βGal formulated with 18 µg pQE30 and 704 induced cellular and humoral responses equivalent to those obtained after vaccination with 100 µg naked DNA or 20 µg pCMV-βGal formulated with 704 (Figure 3a,b). In the absence of carrier DNA, a single injection of 2 µg pCMV-βGal formulated with 704 induced an IFNγ ELISPOT response that peaked at day 21 and was still detectable, though twofold lower, at day 28 (Figure 3e). This response was significantly lower than that observed after vaccination with 20 µg pCMV-βGal formulated with 704 (Figure 3f). Because maximal immune responses to βGal were maintained in the presence of excess pQE30, which does not contain sequences coding for βGal, the effect of carrier DNA was not due to excess βGal expression.

Figure 3.

Figure 3

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High-dose naked DNA vaccination compared to low-dose DNA vaccination with 704 and carrier DNA. (a–d) Groups of mice (n = 5) were injected intramuscularly (i.m.) with 100 µg pCMV-βGal alone (▴), 20 µg pCMV-βGal formulated with 704 (Inline graphic), 2 µg pCMV-βGal + 18 µg pQE30 plasmid formulated with 0.25% 704 (•), or 0.25% 704 alone (▪), and different parameters of the immune response were measured. (a) Humoral response. Geometric mean titers are shown for each group ± standard deviation. The arrow indicates DNA injection at day 0. Figures on the right indicate the pCMV-βGal plasmid dose for each group. For clarity, sera with no detectable βGal-specific IgG are presented at a titer of 1/25. Wide deviations at day 14 and day 21 in the group vaccinated with 2 µg pCMV-βGal + 18 µg pQE30 formulated with 704 are due to two mice that only seroconverted by day 28. (b) Class I–restricted cellular response. Splenocytes were prepared at day 28, stimulated overnight with the βGal ICPMYARV peptide (▴, Inline graphic, •, ▪), or control peptide (▵, ⋄, ○, □), and the number of IFNγ SFCs was determined. Each symbol represents the result from one mouse. (c) Isotype profile of βGal-specific antibodies. βGal-specific IgG1 and IgG2c were titered in sera at day 28 after a single DNA vaccination, and the IgG2c/IgG1 ratio is shown. (d) T-helper response. Splenocytes were prepared at day 28, stimulated for 72 hours with recombinant βGal protein, and IFNγ and IL-4 in the supernatants were determined by ELISA. *P < 0.05 by the Wilcoxon test. (e) Time course of the class I–restricted response. Splenocytes were prepared at 7, 14, 21, and 28 days after a single vaccination with 2 µg pCMV-βGal formulated with 0.25% 704 without carrier DNA, then stimulated overnight with the βGal ICPMYARV peptide (•) or control peptide (○), and the number of IFNγ SFCs was determined. (f) Groups of mice (n = 6) were injected i.m. with 2 µg or 20 µg pCMV-βGal formulated with 704 as indicated. Splenocytes were prepared at day 28, stimulated overnight with the βGal ICPMYARV peptide (•, Inline graphic) or control peptide (○, ⋄), and the number of IFNγ SFCs was determined. Each symbol represents the result from one mouse. *P < 0.05 by the Wilcoxon test. IL-4, interleukin-4; SFC, spot-forming cell.

Moreover, formulation with 704 did not diminish the Th1 bias of the immune response induced by DNA vaccination, as attested by the predominance of IgG2c antibodies (Figure 3c), and the secretion of large amounts of IFNγ, but no detectable interleukin-4 after the stimulation of splenocytes with recombinant βGal protein (Figure 3d). Therefore, formulation of plasmid DNA with 704 allowed a 50-fold reduction in the dose of antigen-coding plasmid, while maintaining the Th1 orientation of i.m. DNA vaccination, on condition that total DNA dose remained at least 20 µg per mouse.

Low-dose DNA vaccination with 704 confers antitumor immunity

The efficacy of low-dose DNA vaccination with 704 was then tested in two tumor vaccination models in transgenic AFP-βGal mice. These animals express βGal protein in the liver during fetal development, but not during adult life.32 They have a degree of central tolerance to βGal, and have potential as a model for tumor vaccines, with βGal as the model tumor antigen.

In the first experiment, groups of AFP-βGal mice were given a single i.m. injection of phosphate-buffered saline (PBS), 109 plaque forming units βGal recombinant serotype 5 adenovirus AdLacZ, or 2 µg pCMV-βGal formulated with 704 and carrier DNA. Two weeks after vaccination, mice were challenged by subcutaneous (s.c.) injection of 5 × 106 Hepa1.6 cells stably expressing βGal (HepaβGal). By 12 days post-challenge, tumors were detected in all mice in the PBS and AdLacZ groups, and in seven of eight mice vaccinated with DNA formulated with 704. Thereafter, tumor growth progressed unchecked in four of the eight mice in the PBS and AdLacZ groups. In contrast, tumors were either eradicated or remained at sizes <150 mm3 in all mice in the DNA plus 704 group (Supplementary Figure S3). Overall, survival was significantly greater in the group receiving DNA formulated with 704 and carrier DNA (P < 0.05, Figure 4a).

Figure 4.

Figure 4

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Efficacy of low-dose DNA vaccination with 704 in Hepa1.6 tumor growth model. (a) Subcutaneous tumor model. Groups (n = 8) of AFP-βGal mice were injected intramuscularly (i.m.) with PBS (•), 109 plaque-forming units βGal recombinant adenovirus AdLacZ (AdβGal, Inline graphic), or 2 µg pCMV-βGal + 18 µg pQE30 formulated with 0.25% 704 (704, ▴). Two weeks after vaccination, mice were challenged subcutaneously with 5 × 106 Hepa1.6 cells stably expressing βGal. Kaplan-Meier analysis of uncontrolled tumor growth (tumor size >250 mm3) in the three groups is shown. Significant differences between groups are indicated. *P < 0.05 by log-rank test. (b–d) Orthotopic tumor model. Groups of AFP-βGal mice were injected i.m. with 0.25% 704 alone (•, n = 6), 100 µg pCMV-βGal (▴, n = 6), or 2 µg pCMV-βGal + 18 µg pQE30 formulated with 0.25% 704 (Inline graphic, n = 6). A booster vaccination was given at day 21, and then at day 28, mice were injected via the portal vein with 2 × 106 HepaβGal cells. Mice were killed 3 weeks later at day 49. (b) Images of livers from two mice in each group. (c) Liver weight as a percentage of body weight. (d) Specific βGal activity in liver extracts. PBS, phosphate-buffered saline.

To confirm these findings, groups of AFP-βGal mice were vaccinated twice (prime day 0, boost day 21) either with 100 µg naked pCMV-βGal (n = 7), 2 µg pCMV-βGal formulated with 704 and carrier DNA (n = 6), or 704 alone (n = 6) before challenge with 2 × 106 HepaβGal cells injected into the portal vein. In this model, orthotopic liver tumors developed 3–5 weeks after challenge in the control group, and there was no evidence of spontaneous tumor rejection. In the experiment shown, all mice were killed 3 weeks after challenge, and tumor growth was assessed by comparing gross liver morphology (Figure 4b), liver weight as a percentage of body weight (Figure 4c), and βGal activity in liver extracts as a specific marker of tumor cell growth (Figure 4d). All six animals in the control group expressed significant amounts of βGal (>1 ng/mg protein, Figure 4d) in the liver, indicating the growth of HepaβGal cells, and this was accompanied by liver hyperplasia (liver weight >10% of body weight, Figure 4c) in five of the six control animals. In contrast, liver morphology and weight remained normal in all vaccinated animals (Figure 4b,c), and only one of the six animals vaccinated with 2 µg pCMV-βGal DNA formulated with 704 and carrier DNA had detectable βGal expression in the liver (Figure 4d). Vaccination with 2 µg pCMV-βGal DNA formulated with 704 and carrier DNA was therefore as effective as vaccination with 100 µg naked pCMV-βGal DNA in a physiologically relevant tumor growth model, in which the antigenic target is expressed as a self-antigen and tumor cells grow in their normal tissue environment.

Low-dose DNA vaccination with 704 against ovalbumin

To extend our findings to a second model antigen, mice were vaccinated either with 20 µg pCMV-Ova formulated with 704, or 2 µg pCMV-Ova formulated with 704 in the presence or absence of 18 µg pQE30. The class I–restricted cellular response was followed by pentamer staining. No ovalbumin (Ova)-specific CD8+ T-cells were detected after a single vaccination, and peak numbers of Ova-specific CD8+ T-cells reached 0.3–0.4% of CD8+ lymphocytes 14 days after a booster vaccination (Figure 5a). Vaccination with 2 µg pCMV-Ova formulated with 704 and carrier DNA gave an equivalent response to that observed after vaccination with 20 µg pCMV-Ova formulated with 704. We therefore tested the efficacy of low-dose pCMV-Ova DNA vaccination in the B16-Ova tumor model. Groups of C57BL/6 mice were vaccinated i.m. either with 100 µg naked pCMV-Ova DNA, or 2 µg pCMV-Ova formulated with 704 and 18 µg pQE30. A booster vaccination was given at day 21, and 7 days later, mice were challenged by s.c. injection of 2.5 × 105 B16-Ova cells. Tumors developed by day 20 post-challenge in all mice in the control group, whereas tumor growth was significantly delayed (P < 0.01, Figure 5b) in both groups of vaccinated mice. In this model, as in the orthotopic liver tumor model, vaccination with 2 µg antigen-coding plasmid DNA formulated with 704 and carrier DNA was as effective as vaccination with 100 µg naked plasmid DNA encoding tumor antigen.

Figure 5.

Figure 5

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Efficacy of low-dose DNA vaccination with 704 with Ova as model antigen. (a) Groups of C57BL/6 mice (n = 6) were injected intramuscularly (i.m.) with 0.25% 704 alone (□), 2 µg pCMV-Ova formulated with 0.25% 704 (•), 2 µg pCMV-Ova + 18 µg pQE30 formulated with 0.25% 704 (Inline graphic), or 20 µg pCMV-Ova formulated with 0.25% 704 (▴). Arrows indicate DNA injection at day 0 and boost at day 21. Heparinized blood was sampled once per week, and Ova-specific CD8+ cells were identified by staining with H2-Kb:SIINFEKL pentamers. Data points represent median values for each group. Significant differences between groups at the time of peak response (day 35) are indicated. *P < 0.05 by analysis of variance and Tukey's least significant difference test. (b) Groups of C57BL/6 mice (n = 8) were vaccinated i.m. either with 0.25% 704 alone (•), 100 µg naked pCMV-Ova DNA (▪), or 2 µg pCMV-Ova formulated with 704 and 18 µg pQE30 (▴). A booster vaccination was given at day 21, and 7 days later, mice were challenged by subcutaneous injection of 2.5 × 105 B16-Ova cells. Kaplan-Meier analysis of uncontrolled tumor growth (tumor size >250 mm3) in the three groups is shown. Significant differences between groups are indicated. **P < 0.01 by log-rank test.

704 does not possess adjuvant properties for recombinant βGal protein

Previous results implied that 704 enhanced the efficiency of DNA vaccination by increasing antigen expression. However, block copolymers have previously been described as adjuvants for protein antigens.33 To test whether 704 also possessed classical immunological adjuvant activity, recombinant βGal protein was used as antigen either alone, adjuvanted with incomplete Freund's adjuvant, or formulated with 704. Intramuscular injection of 100 µg recombinant βGal protein without adjuvant induced a significant humoral response composed primarily of the IgG1 isotype, and a weak (<50 spot-forming cells (SFCs)/million splenocytes above background) βGal-specific ELISPOT response in four out of five vaccinated animals (Figure 6a–c). As expected, incomplete Freund's adjuvant strongly enhanced the βGal-specific antibody response. In contrast, formulation with 704 did not result in any detectable enhancement of humoral or cellular responses to βGal protein and did not modify the isotype profile of cognate antibodies. Therefore, 704 does not possess adjuvant properties for recombinant βGal protein following i.m. injection. As 704 has an MW of 5.5 kd and a PEO content of 40%, these results are consistent with previous work showing that adjuvant activity for protein antigens was maximal for nonionic block copolymers of 10–12 kd with a PEO content <20%.33

Figure 6.

Figure 6

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704 does not act as an adjuvant for recombinant βGal protein. Groups of mice were injected intramuscularly with 100 µg recombinant βGal protein in vehicle (Inline graphic, n = 5), or formulated with 0.25% 704 (▴, n = 5) or subcutaneous 100 µg recombinant βGal protein in IFA (▪, n = 3). (a) Humoral response. Geometric mean titers are shown for each group ± standard deviation. Significantly higher titers after vaccination with βGal protein in IFA are indicated. **P < 0.01 by analysis of variance and Tukey's least significant difference test. (b) Class I–restricted cellular response. Splenocytes were prepared at day 28, stimulated overnight with the βGal ICPMYARV peptide (Inline graphic, ▴, ▪) or control peptide (⋄, ▵, □), and the number of IFNγ SFCs was determined. Each symbol represents the result from one mouse. (c) Isotype profile of βGal-specific antibodies. βGal-specific IgG1 and IgG2c were titered in sera at day 28 after a single vaccination, and the IgG2c/IgG1 ratio is shown. IFA, incomplete Freund's adjuvant; SFC, spot-forming cell.

Carrier DNA enhances antigen expression and cannot be replaced by CpG-rich oligodeoxynucleotides

Vaccination with low doses of pCMV-βGal formulated with 704 was only effective in the presence of excess carrier DNA. Plasmid DNA has immunological adjuvant activity due to the presence of hypomethylated CpG motifs that are specifically recognized by toll-like receptor 9. However, addition of CpG-rich oligodeoxynucleotides, which are also toll-like receptor 9 agonists, to 2 µg pCMV-βGal formulated with 704 was unable to enhance the cellular or the humoral immune response to βGal (Figure 7a).

Figure 7.

Figure 7

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Effect of CpG ODN and carrier DNA on immune responses and antigen expression. (a) Groups of mice were injected intramuscularly (i.m.) with 2 µg pCMV-βGal formulated with 0.25% 704, in the presence of increasing amounts of CpG containing oligodeoxynucleotides (5, 10, or 20 µg, as indicated). Splenocytes were prepared at day 28, stimulated overnight with the ICPMYARV peptide, and the number of IFNγ SFCs was determined. Mean numbers of IFNγ SFC are shown for each group ± standard deviation. For the humoral response, geometric mean titers are shown for each group ± standard deviation. Differences between groups were not significant. (b) Groups of mice were injected i.m. with 1, 2, or 10 µg pCMV-βGal formulated with 0.25% 704, 50 µg naked pCMV-βGal DNA, or 1 µg pCMV-βGal + 9 µg pQE30 formulated with 0.25% 704 as indicated. Doses given are per tibialis anterior muscle. At day 7 after injection, mice were killed, and βGal expression in injected muscle quantified by the Beta-Glo Assay. Bars show mean ± standard deviation βGal (ng per mg total protein) for n = 6 injected muscles for 1 µg pCMV-βGal formulated with 0.25% 704, and n = 8 injected muscles for all other conditions. *P < 0.05, **P < 0.01 by analysis of variance and Tukey's least significant difference test. βGal expression observed after injection of 1 or 2 µg pCMV-βGal formulated with 0.25% 704 was significantly (P < 0.01) lower than that observed for the other three conditions. ODN, oligodeoxynucleotide; SFC, spot-forming cell.

Because the activation of toll-like receptor 9 did not appear to be sufficient to explain the enhancing effect of carrier DNA on low-dose DNA vaccination, we then tested whether the presence of carrier DNA could modify the expression of antigen after plasmid injection. Mice were injected with increasing amounts of pCMV-βGal (1 to 50 µg pCMV-βGal per TA muscle) in the presence or absence of 704 and pQE30 plasmid as carrier DNA, killed at day 7 after injection, and βGal expression in protein extracts from individual muscles was quantified. As expected, formulation with 704 significantly enhanced βGal expression, as injection of 10 µg pCMV-βGal formulated with 704 resulted in 4.6-fold higher βGal expression than injection of 50 µg pCMV-βGal alone (Figure 7b). However, βGal expression fell off sharply at lower doses of pCMV-βGal, with a 400-fold reduction in βGal expression as DNA dose was reduced from 10 to 1 µg. Addition of carrier DNA resulted in a spectacular restoration of reporter gene expression. βGal expression after injection of 1 µg pCMV-βGal plus 9 µg pQE30 formulated with 704 was equivalent to that observed after injection of 50 µg pCMV-βGal alone, and more than 60-fold greater than that observed after injection of 1 µg pCMV-βGal formulated with 704 without carrier DNA (Figure 7b).

Discussion

So far, clinical trials of DNA vaccination have proven disappointing, as the immune responses observed are well below those that can be obtained in the mouse. However, it is worth bearing in mind that i.m. vaccination with naked DNA is also relatively inefficient in mice. Plasmid doses from 25–100 µg per mouse (1–4 mg/kg for a 25-g mouse) are routinely used, whereas in humans, plasmid DNA is administered at doses of 1–5 mg per injection or 0.014–0.07 mg/kg for a 70-kg adult. This is two orders of magnitude less than the effective i.m. DNA vaccine dose in the mouse, so the efficacy of DNA vaccination may need to be improved by ~100-fold for i.m. DNA vaccination to prove effective in humans.

Over the past decade, a wide variety of molecules have been tested in mice for their ability to enhance DNA vaccination, and the more promising vectors have also been investigated in nonhuman primates.22 Complexing DNA with cationic liposomes,34 linear polethylene-imine,35 the block copolymer CRL1005,36 and β-amino ester polymers37,38 augments immune responses to i.m. DNA vaccination. However, efficient DNA vaccination with microgram levels of plasmid has not been reported for any of these chemical adjuvants, which have been shown to enhance immune responses only at DNA doses of at least 10 µg/mouse. In contrast, a single i.m. injection of DNA formulated with poly(lactide-coglycolide)-cetyltrimethylammonium bromide microparticles39 induced CTL and antibody responses at plasmid doses of 1 µg per mouse, and effective priming was observed at doses of 10 to 100 ng/mouse.40 The efficacy of low-dose DNA vaccination that we observed using 704 appears similar to that reported for poly(lactide-coglycolide)-cetyltrimethylammonium bromide.

However, immune responses to low doses of antigen-encoding plasmid formulated with 704 were only observed in the presence of relatively large amounts (20 µg/mouse) of noncoding carrier DNA. Noncoding plasmid DNA can provide a pro-inflammatory “danger signal” necessary for the initiation of a specific immune response to the expressed antigen through recognition of hypomethylated CpG motifs by toll-like receptor 9.29,30 The two plasmids used as carrier DNA contain a similar number of immunostimulatory CpG motifs.31 pUC19 has one optimal GACGTT motif and 13 suboptimal CpG motifs respecting the general formula “purine-purine-CG-pyrimidine-pyrimidine”, whereas pQE30 contains no optimal motifs, and 16 suboptimal motifs. The antigen-encoding pCMV-βGal plasmid, however, contains one optimal and 44 suboptimal CpG motifs. Taking into account the molar ratios of pCMV-βGal, pUC19, and pQE30, the overall dose of immunostimulatory CpG sequences in a mixture of 2 µg pCMV-βGal with 18 µg pUC19 or pQE30 is similar to that contained in 20 µg pCMV-β. The presence of carrier DNA therefore maintained a constant dose of immunostimulatory CpG sequences as the amount of pCMV-βGal injected was reduced from 20 to 2 µg.

However, CpG-rich oligodeoxynucleotides were unable to reproduce the effect of carrier DNA, suggesting that carrier DNA had another role in addition to potentially providing immunological adjuvant activity. Unexpectedly, we found that carrier DNA had a marked effect on antigen expression after plasmid injection. Without carrier DNA, only trace amounts of βGal protein were expressed after injection of 1 µg pCMV-βGal formulated with 704 per TA muscle (Figure 7b), and only weak immune responses were observed in mice vaccinated with this plasmid dose in the absence of carrier DNA (Figure 3). Addition of excess pQE30 restored antigen expression and cognate immune responses to the levels observed after injection of 50 µg naked pCMV-βGal DNA per TA muscle. Although it is probable that carrier DNA also had immunological adjuvant activity, the correlation between antigen expression and observed immune responses suggests that increased antigen expression makes an important contribution to the enhancement of vaccination efficiency by carrier DNA.

How might excess noncoding DNA increase the in vivo transfection efficiency of a separate plasmid? First, formulation of DNA with 704 may be ineffective at lower DNA concentrations, leading to less efficient gene transfer at low DNA doses. However, preliminary data indicate that both the apparent particle size and DNA entrapment in mixtures of 704 and plasmid DNA remained stable as plasmid DNA concentration was reduced from 200 to 20 µg/ml (Supplementary Table S1). Second, degradation of injected DNA is one factor that limits the efficacy of gene transfer in vivo, as significant increases in reporter gene expression can be obtained by coadministration of the nuclease inhibitor aurintricarboxylic acid.41,42,43 It is possible that excess carrier DNA saturates nuclease activity at the injection site, thereby protecting at least some of the antigen-coding plasmid from degradation, facilitating its uptake and ultimately enhancing expression of the encoded gene.

Although low-dose DNA vaccination with 704 and carrier DNA both induced specific humoral and cellular immune responses to βGal or Ova, and conferred protection against challenge with tumor cells expressing the cognate antigen, the mechanism of tumor rejection was not tested in the present work. In mice, injection of CpG-rich oligonucleotides provokes the secretion of type-1 interferons and pro-inflammatory cytokines such as interleukin-1, interleukin-6, and TNFα by activating dendritic cells, macrophages, and natural killer cells.31 As the plasmids we used contained immunostimulatory CpG motifs, it is possible that innate immune responses such as these were responsible for, or at least contributed to, tumor rejection. Indeed, after s.c. challenge with HepaβGal cells, tumor rejection did not correlate with the induction of either the antibody or the class I–restricted βGal-specific response (Supplementary Table S2). Further work will therefore be required to determine the relative roles of innate and adaptive immune responses in the protection afforded by DNA vaccination with 704.

In conclusion, the results presented in this report should prove to be of significant value for the future development of DNA vaccines, the challenge being to reduce the dose of plasmid DNA encoding tumor or pathogen-derived antigens capable of eliciting immune responses in humans. This would simplify the clinical development and reduce the large-scale production costs of DNA vaccines. It is possible that 704 and carrier DNA will represent an important step toward the development of a pharmaceutically acceptable synthetic vaccination formulation because the same DNA carrier would be suitable for any desired antigen-encoding plasmid DNA. If an acceptable safety profile of the 704/carrier DNA formulation was established, it would also reduce the risks of adverse events during trials of future DNA vaccines, as much lower doses of the untested antigen-encoding plasmid would need to be administered. Furthermore, our results imply that combining DNA vaccines for several different antigens in a single injection could be much more effective than giving each DNA vaccine separately. In effect, each antigen-encoding plasmid could serve as a carrier DNA for the others, so that the levels of expressed antigen and the cognate immune response would be greater in a combined vaccine, compared to the same dose of each plasmid given separately. Ultimately, however, it would be desirable to replace carrier DNA with a synthetic substitute, and ongoing experiments in our lab are working toward this goal.

Materials and Methods

Animal procedures. Transgenic AFP/βGal and C57BL/6 mice (Elevage Janvier, Le Genest, France) were housed in conventional conditions. AFP/βGal mice were generated on a C57BL/6 genetic background and have been described previously.32

Mice aged 8–10 weeks were used for all experiments. For i.m. DNA vaccination, mice were anesthetized with etomidate (40 mg/kg, intraperitoneal), then different DNA-polymer formulations were injected into both TA muscles using a U100 microfine syringe (BD Medical, Rungis, France). For protein vaccination, recombinant βGal (Roche, Rosny- sous-Bois, France) was injected i.m. either alone, or formulated with 0.25% 704, or s.c. in incomplete Freund's adjuvant (Sigma, St Quentin Fallavier, France). Two sites were injected per animal, so DNA and protein doses are given as total dose per animal throughout. The βGal recombinant serotype 5 adenovirus (AdLacZ) has been described previously.44 It was produced by the viral vector platform of the UMR INSERM 649, Nantes, and was administered by one i.m. injection of 109 plaque-forming units AdLacZ in 0.9% NaCl. In all cases, the injection volume was 50 µl per injection site.

Serum was collected by retro-orbital bleeds at different time points after vaccination, and spleens were recovered for analysis of the cellular immune response at the end of each experiment.

Plasmid preparation and formulation. The pCMV-βGal plasmid (Clontech, St Germain en Laye, France) coding for βGal controlled by the human cytomegalovirus immediate-early gene promoter was used as antigen. pCMV-Ova was generously supplied by Dr M.S. Dai (INSERM CIC-04, Nantes, France). It was prepared by sub-cloning the Ova cDNA from a previously described bacterial expression plasmid45 into pCDNA3.1. The cloning vectors pUC19 and pQE30 (Qiagen, Courtaboeuf, France) were used as carrier DNA. All plasmids were purified using EndoFree plasmid purification columns (Qiagen) and were confirmed to be free of endotoxin contamination (endotoxin <0.1 EU/µg plasmid DNA) by the Limulus amoebocyte lysate assay (Lonza, Clermont-Ferrand, France).

The triblock copolymers Lutrol (MW 8,600) and PE6400 (MW 2,900) were supplied by BASF (Levallois, France). The tetrafunctional block copolymers 304 (MW 1,650), 704 (MW 5,500), and 904 (MW 6,600) were kindly supplied by In-Cell-Art (Nantes, France). Plasmid DNA was formulated immediately prior to i.m. injection as previously described.25,26,27,28 Final polymer concentrations were as follows: PE6400 0.05%, Lutrol 3%, 304 5%, 704 0.25%, and 904 0.1%. The CpG containing olideoxynucleotide 1826 (sequence TCC ATG ACG TTC CTG ACG TT) was supplied by Eurogentec (Angers, France).

βGal expression. To visualize βGal expression in injected muscles, TA muscles were dissected, rapidly rinsed in PBS and fixed for 30–60 minutes in 2% paraformaldehyde. Muscles were then washed four times in PBS and stained for LacZ in X-Gal solution (2 mmol/l MgCl2, 4 mmol/l potassium ferricyanide, 4 mmol/l potassium ferrocyanide in PBS, containing X-Gal at 0.4 mg/ml). βGal expression was quantified in muscle extracts using the Beta-Glo Assay System (Promega, Charbonnières, France) according to the manufacturer's protocol.

Measurement of the immune response. Humoral immune responses were measured by enzyme-linked immunosorbent assay. Briefly, 96-well plates (Nunc Maxisorp, Roskilde, Denmark) were coated overnight at 4 °C with 5 µg/ml recombinant βGal in 50 mmol/l NaHCO3 pH 9.5, then blocked for 1 hour at room temperature with PBS 0.05% Tween-20 1% bovine serum albumin before distributing diluted sera in triplicate. Plates were incubated at 37 °C for 90 minutes, then βGal-specific IgG was detected using peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Newmarket, UK) diluted 1/5,000, goat anti-mouse IgG1 (AbD Serotec, Oxford, UK) diluted 1/5,000, or goat anti-mouse IgG2c (AbD Serotec) diluted 1/10,000 in PBS 0.05% Tween-20 1% bovine serum albumin. Plates were washed three times in PBS 0.05% Tween-20 between steps, and peroxidase activity was revealed with 1 mg/ml ortho-phenylene diamine in pH5 citrate buffer. Reactions were stopped by addition of 1 mol/l H2SO4, then absorption was measured at 492 nm. Sera were tested at 1/100, 1/1,000, and 1/10,000, and titers were calculated with respect to doubling dilutions of a control serum present in each enzyme-linked immunosorbent assay plate. Results are presented as log10 titers, which correspond to the log of the last serum dilution that gave an enzyme-linked immunosorbent assay reading greater than the threshold value (optical density > 0.4).

Class I–restricted IFNγ secretion was determined by ELISPOT (Diaclone, Besançon, France), as a marker for the presence of βGal-specific CTL. The H2-Kb restricted ICPMYARV peptide (βGal 497–504 [ref. 46]) was used as a representative βGal epitope. The negative control was either the SIINFEKL peptide (Ova 257–264) or the KRWIILGLNK peptide (HIV gag 263–272). Live splenocytes were counted on a hemocytometer slide by Trypan blue exclusion, resuspended at 1 × 106/ml in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/l L-glutamine, penicillin, and streptomycin—all from Invitrogen, Paisley, UK), then distributed in triplicate at 1 × 105 cells/well. Cells were incubated overnight at 37 °C and 5% CO2 in the presence of 5 µg/ml Concanavalin A or 4 µg/ml peptide. SFCs were detected according to the manufacturer's protocol, automatically counted on an AID ELISPOT reader (Autoimmun Diagnostika, Strassberg, Germany) and results expressed as SFC/million splenocytes. To correct for cell counting errors, peptide-specific SFC counts from the n th mouse were normalized by multiplying by (Mean ConA stimulated SFC over all wells/ConA stimulated SFC from the n th mouse). Normalization was consistently found to reduce the within-group variance.

To detect βGal-specific cytokine secretion, splenocytes were cultured at 5 × 106 cells/ml in complete medium. Recombinant βGal was added at 20 µg/ml, and cells were incubated at 37 °C and 5% CO2. Supernatants were harvested at 72 hours, then interleukin-4 and IFNγ concentrations were determined using sandwich enzyme-linked immunosorbent assay kits (Diaclone) according to the manufacturer's protocol.

Ova-specific CD8+ T-cell responses were measured by flow cytometry after staining peripheral blood leukocytes with PE-conjugated Pro5 H2-Kb:SIINFEKL pentamers and FITC-conjugated anti-CD8 (Proimmune, Oxford, UK) according to the manufacturer's protocol.

Tumor vaccination model. Tumor cells were grown to 50–70% confluence before trypsinization, rinsed at least three times in PBS and injected into mice 1 week after the last immunization.

For the s.c. tumor models, AFP/βGal and C57BL/6 mice were injected in the left flank with 5 × 106 HepaβGal cells or with 2.5 × 105 B16-Ova cells, respectively, in 50 µl of serum-free Dulbecco's modified Eagle's medium. Tumor development was monitored by measuring the size of the tumor twice a week. Tumor volume was calculated as follows: V = length × width2 × 0.52. Mice were euthanized when tumors reached 2 cm in diameter.

In the orthotopic liver tumor model, AFP/βGal mice were subjected to laparotomy and were injected via the portal vein with 2 × 106 HepaβGal cells in 100 µl of serum-free DMEM. The injection site was covered by a cotton ball to stop bleeding before closing the abdominal cavity. Mice were killed 3 weeks later, livers were weighed, and βGal enzymatic activity was determined as described.32

Statistical methods. Quantitative differences in anti-βGal titer, βGal-specific IFNγ SFC, and levels of cytokine secretion were tested by one-way analysis of variance, followed by Tukey's least significant difference post hoc test. Statistical tests were performed on log titers because the values are measured on an exponential scale. ELISPOT data were consistently right-skewed, so a log-transform was used in order to apply parametric tests, such as analysis of variance, that require a normal distribution. Differences in the survival times of mice after tumor cell injection were tested by the log-rank test. Statistical tests were performed using the following web resources:

  http://bayes.math.montana.edu/cgi-bin/Rweb/buildModules.cgi for analysis of variance,

  http://www.fon.hum.uva.nl/Service/Statistics/Wilcoxon_Test.html for the Wilcoxon test,

  http://department.obg.cuhk.edu.hk/researchsupport/statstesthome.asp for Tukey's least significant difference, and

  http://bioinf.wehi.edu.au/software/russell/logrank/ for the log-rank test.

SUPPLEMENTARY MATERIALFigure S1. Transgene expression after i.m. injection of pCLuc formulated with different concentrations of 704. Groups of Swiss mice were injected i.m. with 15 μg pCMV-luc, coding for the luciferase reporter gene controlled by the human CMV immediate early gene promoter, formulated with different concentrations of 704 as indicated. Seven days after plasmid injection, tibial anterior (TA) muscles were dissected, frozen in liquid nitrogen and homogenized in 1 mL of Reporter Lysis Buffer (Promega) supplemented with a protease inhibitor cocktail (Roche Diagnostics). After centrifugation at 10,000 rpm for 4 min, luciferase activity was measured from an aliquot of supernatant with Victor2 (PerkinElmer), using a Luciferase Assay System (Promega). Each muscle extract was analyzed in duplicate, and 6 TA muscles were analyzed per condition. Luciferase activity was determined by measuring the light emission after addition of 100 μL of luciferase assay substrate to 10 μL of supernatant. Mean number of counts per second (cps) ± SEM are shown.Figure S2. Dose response of DNA vaccination with 0.25% 704. Groups of mice were injected i.m. with 100 μg (▴), 20 μg (⧫), 4 μg (•) pCMV-βGal formulated with 0.25% 704, or vehicle (□). n=4 for all groups, except injection with vehicle only, n=3. (A) Humoral response. Geometric mean titres (GMT) are shown for each group ± standard deviation. Arrows indicate DNA injection at d0 and boost at d21. Figures on the right indicate the plasmid DNA dose per mouse for each group. (B) Class-I restricted cellular response. Splenocytes were prepared at d42, stimulated overnight with the ICPMYARV peptide, and the number of IFN-γ SFC was determined. Each symbol represents the result from one mouse. Significant differences between groups are indicated. * p<0.05 by ANOVA and Tukey's LSD post-hoc test.Figure S3. Tumor Growth curves after s.c. injection of Hepa-βGal cells. Groups (n=8) of AFP-βGal mice were injected i.m. with PBS (•), 109 pfu βGal recombinant adenovirus AdLacZ (AdβGal, ⧫), or 2 μg pCMV-βGal + 18 μg pQE30 formulated with 0.25% 704 (704, ▴). Two weeks after vaccination, mice were challenged s.c. with 5x106 Hepa1.6 cells stably expressing βGal. Tumor size was measured twice a week.Table S1. Physico-chemical characterisation of DNA/704 complexes.Table S2. Parameters of the βGal-specific Immune response in mice challenged with HepaβGal tumor cells s.cut.

Supplementary Material

Figure S1.

Transgene expression after i.m. injection of pCLuc formulated with different concentrations of 704. Groups of Swiss mice were injected i.m. with 15 μg pCMV-luc, coding for the luciferase reporter gene controlled by the human CMV immediate early gene promoter, formulated with different concentrations of 704 as indicated. Seven days after plasmid injection, tibial anterior (TA) muscles were dissected, frozen in liquid nitrogen and homogenized in 1 mL of Reporter Lysis Buffer (Promega) supplemented with a protease inhibitor cocktail (Roche Diagnostics). After centrifugation at 10,000 rpm for 4 min, luciferase activity was measured from an aliquot of supernatant with Victor2 (PerkinElmer), using a Luciferase Assay System (Promega). Each muscle extract was analyzed in duplicate, and 6 TA muscles were analyzed per condition. Luciferase activity was determined by measuring the light emission after addition of 100 μL of luciferase assay substrate to 10 μL of supernatant. Mean number of counts per second (cps) ± SEM are shown.

Click here for supplemental data (76.1KB, tiff)
Figure S2.

Dose response of DNA vaccination with 0.25% 704. Groups of mice were injected i.m. with 100 μg (▴), 20 μg (⧫), 4 μg (•) pCMV-βGal formulated with 0.25% 704, or vehicle (□). n=4 for all groups, except injection with vehicle only, n=3. (A) Humoral response. Geometric mean titres (GMT) are shown for each group ± standard deviation. Arrows indicate DNA injection at d0 and boost at d21. Figures on the right indicate the plasmid DNA dose per mouse for each group. (B) Class-I restricted cellular response. Splenocytes were prepared at d42, stimulated overnight with the ICPMYARV peptide, and the number of IFN-γ SFC was determined. Each symbol represents the result from one mouse. Significant differences between groups are indicated. * p<0.05 by ANOVA and Tukey's LSD post-hoc test.

Click here for supplemental data (79KB, tiff)
Figure S3.

Tumor Growth curves after s.c. injection of Hepa-βGal cells. Groups (n=8) of AFP-βGal mice were injected i.m. with PBS (•), 109 pfu βGal recombinant adenovirus AdLacZ (AdβGal, ⧫), or 2 μg pCMV-βGal + 18 μg pQE30 formulated with 0.25% 704 (704, ▴). Two weeks after vaccination, mice were challenged s.c. with 5x106 Hepa1.6 cells stably expressing βGal. Tumor size was measured twice a week.

Click here for supplemental data (83.2KB, tiff)
Table S1.

Physico-chemical characterisation of DNA/704 complexes.

Click here for supplemental data (28KB, doc)
Table S2.

Parameters of the βGal-specific Immune response in mice challenged with HepaβGal tumor cells s.cut.

Click here for supplemental data (39KB, doc)

Acknowledgments

We thank Béhazine Combadière (INSERM UMR 543, Paris, France) for advice on analysis of Ova-specific immune responses, Nicolas Ferry (INSERM UMR 948, Nantes, France) for useful discussions throughout the work, and Marie-Aude Muller and Pascal Gervier (CNRS UMR 6204, Nantes, France) for animal maintenance. This work was supported by grants from the Association Française contre les Myopathies (Evry, France), Vaincre la Mucoviscidose (Paris, France), the Association pour la Recherche sur le Cancer (Villejuif, France), and the Ligue Contre le Cancer (Departmental Committees 29, 35, 44, and 49, France). J.C. was the recipient of a fellowship from the Pays de la Loire region.

REFERENCES

  1. Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993;259:1745–1749. doi: 10.1126/science.8456302. [DOI] [PubMed] [Google Scholar]
  2. Raz E, Tighe H, Sato Y, Corr M, Dudler JA, Roman M, et al. Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization. Proc Natl Acad Sci USA. 1996;93:5141–5145. doi: 10.1073/pnas.93.10.5141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Donnelly JJ, Ulmer JB, Shiver JW., and , Liu MA. DNA vaccines. Annu Rev Immunol. 1997;15:617–648. doi: 10.1146/annurev.immunol.15.1.617. [DOI] [PubMed] [Google Scholar]
  4. Wang R, Epstein J, Baraceros FM, Gorak EJ, Charoenvit Y, Carucci DJ, et al. Induction of CD4(+) T cell-dependent CD8(+) type 1 responses in humans by a malaria DNA vaccine. Proc Natl Acad Sci USA. 2001;98:10817–10822. doi: 10.1073/pnas.181123498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boyer JD, Cohen AD, Vogt S, Schumann K, Nath B, Ahn L, et al. Vaccination of seronegative volunteers with a human immunodeficiency virus type 1 env/rev DNA vaccine induces antigen-specific proliferation and lymphocyte production of β-chemokines. J Infect Dis. 2000;181:476–483. doi: 10.1086/315229. [DOI] [PubMed] [Google Scholar]
  6. Timmerman JM, Singh G, Hermanson G, Hobart P, Czerwinski DK, Taidi B, et al. Immunogenicity of a plasmid DNA vaccine encoding chimeric idiotype in patients with β-cell lymphoma. Cancer Res. 2002;62:5845–5852. [PubMed] [Google Scholar]
  7. Rice J, Buchan S., and , Stevenson FK. Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen. J Immunol. 2002;169:3908–3913. doi: 10.4049/jimmunol.169.7.3908. [DOI] [PubMed] [Google Scholar]
  8. Stevenson FK, Ottensmeier CH, Johnson P, Zhu D, Buchan SL, McCann KJ, et al. DNA vaccines to attack cancer Proc Natl Acad Sci USA 200410114646–14652.Suppl 2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hauser H, Shen L, Gu QL, Krueger S., and , Chen SY. Secretory heat-shock protein as a dendritic cell-targeting molecule: a new strategy to enhance the potency of genetic vaccines. Gene Ther. 2004;11:924–932. doi: 10.1038/sj.gt.3302160. [DOI] [PubMed] [Google Scholar]
  10. Rapp UK., and , Kaufmann SH. DNA vaccination with gp96-peptide fusion proteins induces protection against an intracellular bacterial pathogen. Int Immunol. 2004;16:597–605. doi: 10.1093/intimm/dxh064. [DOI] [PubMed] [Google Scholar]
  11. Schirmbeck R, Riedl P, Kupferschmitt M, Wegenka U, Hauser H, Rice J, et al. Priming protective CD8 T cell immunity by DNA vaccines encoding chimeric, stress protein-capturing tumor-associated antigen. J Immunol. 2006;177:1534–1542. doi: 10.4049/jimmunol.177.3.1534. [DOI] [PubMed] [Google Scholar]
  12. Ulmer JB, DeWitt CM, Chastain M, Friedman A, Donnelly JJ, McClements WL, et al. Enhancement of DNA vaccine potency using conventional aluminum adjuvants. Vaccine. 1999;18:18–28. doi: 10.1016/s0264-410x(99)00151-6. [DOI] [PubMed] [Google Scholar]
  13. Lodmell DL, Ray NB, Ulrich JT., and , Ewalt LC. DNA vaccination of mice against rabies virus: effects of the route of vaccination and the adjuvant monophosphoryl lipid A (MPL) Vaccine. 2000;18:1059–1066. doi: 10.1016/s0264-410x(99)00352-7. [DOI] [PubMed] [Google Scholar]
  14. Calarota SA., and , Weiner DB. Enhancement of human immunodeficiency virus type 1-DNA vaccine potency through incorporation of T-helper 1 molecular adjuvants. Immunol Rev. 2004;199:84–99. doi: 10.1111/j.0105-2896.2004.00150.x. [DOI] [PubMed] [Google Scholar]
  15. Kim JJ, Trivedi NN, Nottingham LK, Morrison L, Tsai A, Hu Y, et al. Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens. Eur J Immunol. 1998;28:1089–1103. doi: 10.1002/(SICI)1521-4141(199803)28:03<1089::AID-IMMU1089>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  16. Haddad D, Ramprakash J, Sedegah M, Charoenvit Y, Baumgartner R, Kumar S, et al. Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cells into injected muscles. J Immunol. 2000;165:3772–3781. doi: 10.4049/jimmunol.165.7.3772. [DOI] [PubMed] [Google Scholar]
  17. Sumida SM, McKay PF, Truitt DM, Kishko MG, Arthur JC, Seaman MS, et al. Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines. J Clin Invest. 2004;114:1334–1342. doi: 10.1172/JCI22608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Iga M, Boissonnas A, Mahé B, Bonduelle O, Combadière C., and , Combadière B. Single CX3CL1-Ig DNA administration enhances T cell priming in vivo. Vaccine. 2007;25:4554–4563. doi: 10.1016/j.vaccine.2007.04.028. [DOI] [PubMed] [Google Scholar]
  19. Deml L, Bojak A, Steck S, Graf M, Wild J, Schirmbeck R, et al. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol. 2001;75:10991–11001. doi: 10.1128/JVI.75.22.10991-11001.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Steinberg T, Ohlschläger P, Sehr P, Osen W., and , Gissmann L. Modification of HPV 16 E7 genes: correlation between the level of protein expression and CTL response after immunization of C57BL/6 mice. Vaccine. 2005;23:1149–1157. doi: 10.1016/j.vaccine.2004.08.027. [DOI] [PubMed] [Google Scholar]
  21. van Drunen Littel-van den Hurk S, Babiuk SL., and , Babiuk LA. Strategies for improved formulation and delivery of DNA vaccines to veterinary target species. Immunol Rev. 2004;199:113–125. doi: 10.1111/j.0105-2896.2004.00140.x. [DOI] [PubMed] [Google Scholar]
  22. Greenland JR., and , Letvin NL. Chemical adjuvants for plasmid DNA vaccines. Vaccine. 2007;25:3731–3741. doi: 10.1016/j.vaccine.2007.01.120. [DOI] [PubMed] [Google Scholar]
  23. Zhang HY, Sun SH, Guo YJ, Chen ZH, Huang L, Gao YJ, et al. Tissue distribution of a plasmid DNA containing epitopes of foot-and-mouth disease virus in mice. Vaccine. 2005;23:5632–5640. doi: 10.1016/j.vaccine.2005.06.029. [DOI] [PubMed] [Google Scholar]
  24. Kabanov A, Zhu J., and , Alakhov V. Pluronic block copolymers for gene delivery. Adv Genet. 2005;53PA:231–261. doi: 10.1016/S0065-2660(05)53009-8. [DOI] [PubMed] [Google Scholar]
  25. Pitard B, Pollard H, Agbulut O, Lambert O, Vilquin JT, Cherel Y, et al. A nonionic amphiphile agent promotes gene delivery in vivo to skeletal and cardiac muscles. Hum Gene Ther. 2002;13:1767–1775. doi: 10.1089/104303402760293592. [DOI] [PubMed] [Google Scholar]
  26. Desigaux L, Gourden C, Bello-Roufaï M, Richard P, Oudrhiri N, Lehn P, et al. Nonionic amphiphilic block copolymers promote gene transfer to the lung. Hum Gene Ther. 2005;16:821–829. doi: 10.1089/hum.2005.16.821. [DOI] [PubMed] [Google Scholar]
  27. Pitard B, Bello-Roufaï M, Lambert O, Richard P, Desigaux L, Fernandes S, et al. Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer. Nucleic Acids Res. 2004;32:e159. doi: 10.1093/nar/gnh153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Richard P, Bossard F, Desigaux L, Lanctin C, Bello-Roufai M., and , Pitard B. Amphiphilic block copolymers promote gene delivery in vivo to pathological skeletal muscles. Hum Gene Ther. 2005;16:1318–1324. doi: 10.1089/hum.2005.16.1318. [DOI] [PubMed] [Google Scholar]
  29. Roman M, Martin-Orozco E, Goodman JS, Nguyen MD, Sato Y, Ronaghy A, et al. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat Med. 1997;3:849–854. doi: 10.1038/nm0897-849. [DOI] [PubMed] [Google Scholar]
  30. Ishii KJ., and , Akira S. Innate immune recognition of, and regulation by, DNA. Trends Immunol. 2006;27:525–532. doi: 10.1016/j.it.2006.09.002. [DOI] [PubMed] [Google Scholar]
  31. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol. 2002;20:709–760. doi: 10.1146/annurev.immunol.20.100301.064842. [DOI] [PubMed] [Google Scholar]
  32. Cany J, Avril A, Pichard V, Aubert D, Ferry N., and , Conchon S. A transgenic mouse with β-galactosidase as a fetal liver self-antigen for immunotherapy studies. J Hepatol. 2007;47:396–403. doi: 10.1016/j.jhep.2007.03.018. [DOI] [PubMed] [Google Scholar]
  33. Newman MJ, Todd CW., and , Balusubramanian M. Design and development of adjuvant-active nonionic block copolymers. J Pharm Sci. 1998;87:1357–1362. doi: 10.1021/js980072c. [DOI] [PubMed] [Google Scholar]
  34. Perrie Y, Frederik PM., and , Gregoriadis G. Liposome-mediated DNA vaccination: the effect of vesicle composition. Vaccine. 2001;19:3301–3310. doi: 10.1016/s0264-410x(00)00432-1. [DOI] [PubMed] [Google Scholar]
  35. Bos GW, Kanellos T, Crommelin DJ, Hennink WE., and , Howard CR. Cationic polymers that enhance the performance of HbsAg DNA in vivo. Vaccine. 2004;23:460–469. doi: 10.1016/j.vaccine.2004.06.020. [DOI] [PubMed] [Google Scholar]
  36. Casimiro DR, Chen L, Fu TM, Evans RK, Caulfield MJ, Davies ME, et al. Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene. J Virol. 2003;77:6305–6313. doi: 10.1128/JVI.77.11.6305-6313.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Little SR, Lynn DM, Ge Q, Anderson DG, Puram SV, Chen J, et al. Poly-β amino ester-containing microparticles enhance the activity of nonviral genetic vaccines. Proc Natl Acad Sci USA. 2004;101:9534–9539. doi: 10.1073/pnas.0403549101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Greenland JR, Liu H, Berry D, Anderson DG, Kim WK, Irvine DJ, et al. β-amino ester polymers facilitate in vivo DNA transfection and adjuvant plasmid DNA immunization. Mol Ther. 2005;12:164–170. doi: 10.1016/j.ymthe.2005.01.021. [DOI] [PubMed] [Google Scholar]
  39. Singh M, Briones M, Ott G., and , O'Hagan D. Cationic microparticles: a potent delivery system for DNA vaccines. Proc Natl Acad Sci USA. 2000;97:811–816. doi: 10.1073/pnas.97.2.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. O'Hagan D, Singh M, Ugozzoli M, Wild C, Barnett S, Chen M, et al. Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines. J Virol. 2001;75:9037–9043. doi: 10.1128/JVI.75.19.9037-9043.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Glasspool-Malone J, Steenland PR, McDonald RJ, Sanchez RA, Watts TL, Zabner J, et al. DNA transfection of macaque and murine respiratory tissue is greatly enhanced by use of a nuclease inhibitor. J Gene Med. 2002;4:323–322. doi: 10.1002/jgm.259. [DOI] [PubMed] [Google Scholar]
  42. Niedzinski EJ, Chen YJ, Olson DC, Parker EA, Park H, Udove JA, et al. Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation. Gene Ther. 2003;10:2133–2138. doi: 10.1038/sj.gt.3302125. [DOI] [PubMed] [Google Scholar]
  43. Walther W, Stein U, Siegel R, Fichtner I., and , Schlag PM. Use of the nuclease inhibitor aurintricarboxylic acid (ATA) for improved non-viral intratumoral in vivo gene transfer by jet-injection. J Gene Med. 2005;7:477–485. doi: 10.1002/jgm.690. [DOI] [PubMed] [Google Scholar]
  44. Arbuthnot PB, Bralet MP, Le Jossic C, Dedieu JF, Perricaudet M, Bréchot C, et al. In vitro and in vivo hepatoma cell-specific expression of a gene transferred with an adenoviral vector. Hum Gene Ther. 1996;7:1503–1514. doi: 10.1089/hum.1996.7.13-1503. [DOI] [PubMed] [Google Scholar]
  45. Radford KJ, Higgins DE, Pasquini S, Cheadle EJ, Carta L, Jackson AM, et al. A recombinant E. coli vaccine to promote MHC class I-dependent antigen presentation: application to cancer immunotherapy. Gene Ther. 2002;9:1455–1463. doi: 10.1038/sj.gt.3301812. [DOI] [PubMed] [Google Scholar]
  46. Oukka M, Cohen-Tannoudji M, Tanaka Y, Babinet C., and , Kosmatopoulos K. Medullary thymic epithelial cells induce tolerance to intracellular proteins. J Immunol. 1996;156:968–975. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Transgene expression after i.m. injection of pCLuc formulated with different concentrations of 704. Groups of Swiss mice were injected i.m. with 15 μg pCMV-luc, coding for the luciferase reporter gene controlled by the human CMV immediate early gene promoter, formulated with different concentrations of 704 as indicated. Seven days after plasmid injection, tibial anterior (TA) muscles were dissected, frozen in liquid nitrogen and homogenized in 1 mL of Reporter Lysis Buffer (Promega) supplemented with a protease inhibitor cocktail (Roche Diagnostics). After centrifugation at 10,000 rpm for 4 min, luciferase activity was measured from an aliquot of supernatant with Victor2 (PerkinElmer), using a Luciferase Assay System (Promega). Each muscle extract was analyzed in duplicate, and 6 TA muscles were analyzed per condition. Luciferase activity was determined by measuring the light emission after addition of 100 μL of luciferase assay substrate to 10 μL of supernatant. Mean number of counts per second (cps) ± SEM are shown.

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Figure S2.

Dose response of DNA vaccination with 0.25% 704. Groups of mice were injected i.m. with 100 μg (▴), 20 μg (⧫), 4 μg (•) pCMV-βGal formulated with 0.25% 704, or vehicle (□). n=4 for all groups, except injection with vehicle only, n=3. (A) Humoral response. Geometric mean titres (GMT) are shown for each group ± standard deviation. Arrows indicate DNA injection at d0 and boost at d21. Figures on the right indicate the plasmid DNA dose per mouse for each group. (B) Class-I restricted cellular response. Splenocytes were prepared at d42, stimulated overnight with the ICPMYARV peptide, and the number of IFN-γ SFC was determined. Each symbol represents the result from one mouse. Significant differences between groups are indicated. * p<0.05 by ANOVA and Tukey's LSD post-hoc test.

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Figure S3.

Tumor Growth curves after s.c. injection of Hepa-βGal cells. Groups (n=8) of AFP-βGal mice were injected i.m. with PBS (•), 109 pfu βGal recombinant adenovirus AdLacZ (AdβGal, ⧫), or 2 μg pCMV-βGal + 18 μg pQE30 formulated with 0.25% 704 (704, ▴). Two weeks after vaccination, mice were challenged s.c. with 5x106 Hepa1.6 cells stably expressing βGal. Tumor size was measured twice a week.

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Table S1.

Physico-chemical characterisation of DNA/704 complexes.

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Table S2.

Parameters of the βGal-specific Immune response in mice challenged with HepaβGal tumor cells s.cut.

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Articles from Molecular Therapy: the Journal of the American Society of Gene Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

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