Volume 17, Issue 8, Pages 1473-1481 (August 2009) DNA/Amphiphilic Block Copolymer Nanospheres Promote Low-dose DNA Vaccination  Dorian McIlroy, Benoît Barteau, Jeannette Cany, Peggy Richard, Clothilde Gourden, Sophie Conchon, Bruno Pitard  Molecular Therapy  Volume 17, Issue 8, Pages 1473-1481 (August 2009) DOI: 10.1038/mt.2009.84 Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 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 (♦) 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. Molecular Therapy 2009 17, 1473-1481DOI: (10.1038/mt.2009.84) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Effects of carrier plasmid on DNA vaccination with 704. Groups of mice (n = 4) were injected intramuscularly with 20 µg (▴, ▵), 2 µg (♦, ⋄), 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 (▴, ♦, •) 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. Molecular Therapy 2009 17, 1473-1481DOI: (10.1038/mt.2009.84) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 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 (♦), 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 (▴, ♦, •, ▪), 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 (•, ♦) 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. Molecular Therapy 2009 17, 1473-1481DOI: (10.1038/mt.2009.84) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 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, ♦), 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 (♦, 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. Molecular Therapy 2009 17, 1473-1481DOI: (10.1038/mt.2009.84) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 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 (♦), 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. Molecular Therapy 2009 17, 1473-1481DOI: (10.1038/mt.2009.84) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 704 does not act as an adjuvant for recombinant βGal protein. Groups of mice were injected intramuscularly with 100 µg recombinant βGal protein in vehicle (♦, 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 (♦, ▴, ▪) 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. Molecular Therapy 2009 17, 1473-1481DOI: (10.1038/mt.2009.84) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 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. Molecular Therapy 2009 17, 1473-1481DOI: (10.1038/mt.2009.84) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions