Enhancing DNA vaccine potency by combining a strategy to prolong dendritic cell life and intracellular targeting strategies with a strategy to boost CD4+ T cell

Abstract

Intradermal administration of DNA vaccines, using a gene gun, represents an effective means of delivering DNA directly into professional antigen-presenting cells (APCs) in the skin and thus allows the application of strategies to modify the properties of APCs to enhance DNA vaccine potency. In the current study, we hypothesized that the potency of human papillomavirus (HPV) type 16 E7 DNA vaccines employing intracellular targeting strategies combined with a strategy to prolong the life of dendritic cells (DCs) could be further enhanced by the addition of a DNA vaccine capable of generating high numbers of pan-HLA-DR reactive epitope (PADRE)-specific CD4(+) T cells. We observed that the addition of PADRE DNA to E7 DNA vaccines employing intracellular targeting strategies with a strategy to prolong the life of DCs led to significant enhancement of E7-specific CD8(+) effector and memory T cells as well as significantly improved therapeutic effects against established E7-expressing tumors in tumor-challenged mice. Our data suggest that the potency of a DNA vaccine combining an intracellular targeting strategy as well as a strategy to prolong the life of DCs can be further enhanced by addition of DNA that is capable of generating high numbers of PADRE-specific CD4(+) T cells.

FIG. 1

Flow cytometric analysis of E7-specific CD8⁺ T cells in mice vaccinated with various DNA vaccines. C57BL/6 mice (five per group) were immunized, at 1.98 μg of DNA per mouse, with various DNA vaccine mixtures (listed in Table 1) twice with a 1-week interval. Splenocytes from vaccinated mice were harvested 1 week after the last vaccination and characterized for E7-specific CD8⁺ T cells, using intracellular IFN-γ staining followed by flow cytometric analysis. (A) Representative flow cytometric data. Numbers in the upper right-hand corners represent the number of E7-specific IFN-γ-secreting CD8⁺ T cells per 5 × 10⁶ pooled splenocytes. (B) Bar graph depicting numbers of E7-specific IFN-γ-secreting CD8⁺ T cells per 3 × 10⁵ pooled splenocytes (means + SD). Data are from one representative experiment of two performed.

FIG. 2

In vivo treatment experiments. C57BL/6 mice (five per group) were first challenged by subcutaneous injection with TC1 tumor cells (5 × 10⁴/mouse). Three days after tumor challenge, the mice were administered, at 1.98 μg of DNA per mouse, various DNA vaccine mixtures (listed in Table 1) three times with 4-day intervals. The mice were monitored for evidence of tumor growth by inspection and palpitation twice per week. Tumor volume was measured starting from day 7 after tumor challenge. (A) Line graph depicting tumor volume in mice in the tumor treatment experiments (means ± SE). (B) Kaplan–Meier survival analysis of mice in the tumor treatment experiments. Data shown are from one representative experiment of two performed.

FIG. 3

Flow cytometric analysis of E7-specific CD8⁺ T cells in mice vaccinated with various DNA vaccine combinations 2 months after vaccination. C57BL/6 mice (five per group) were immunized, at 1.98 μg of DNA per mouse, with various DNA vaccine mixtures (listed in Table 1) twice with a 1-week interval. Splenocytes from vaccinated mice were harvested 2 months after the last vaccination and characterized for E7-specific CD8⁺ T cells, using intracellular IFN-γ staining followed by flow cytometric analysis. (A) Representative flow cytometric data. Numbers in the upper right-hand corner represent the number of E7-specific IFN-γ-secreting CD8⁺ T cells per 5 ×10⁶ pooled splenocytes 2 months after the last vaccination. (B) Bar graph depicting the numbers of E7-specific IFN-γ-secreting CD8⁺ T cells per 3 × 10⁵ pooled splenocytes 2 months after the last vaccination (means + SD). Data are from one representative experiment of two performed.

FIG. 4

Long-term in vivo tumor protection experiments. C57BL/6 mice (five per group) were immunized, at 1.98 μg of DNA per mouse, with various DNA vaccine mixtures (listed in Table 1) twice with a 1-week interval. Two months after the last vaccination, the mice were challenged by subcutaneous injection of TC-1 cells (3 × 10⁵/mouse). The mice were monitored for evidence of tumor growth by inspection and palpitation twice per week. Tumor volume was measured starting from day 7 after tumor challenge. (A) Line graph depicting tumor volume in mice challenged with TC-1 cells (means ± SE). (B) Kaplan–Meier survival analysis of mice challenged with TC-1 cells. Data are from one representative experiment of two performed.

FIG. 5

Flow cytometric analysis of E7-specific CD8⁺ T cells in DNA-vaccinated mice. C57BL/6 mice (five per group) were immunized, at 1.98 μg of DNA per mouse, with various DNA vaccine mixtures (listed in Table 2) twice with a 1-week interval. Splenocytes from vaccinated mice were harvested 1 week after the last vaccination and characterized for E7-specific CD8⁺ T cells, using intracellular IFN-γ staining followed by flow cytometric analysis. (A) Representative flow cytometric data. Numbers in the upper right-hand corners represent the number of E7-specific IFN-γ-secreting CD8⁺ T cells per 5 × 10⁶ pooled splenocytes. (B) Bar graph depicting the numbers of E7-specific IFN-γ-secreting CD8⁺ T cells per 3 × 10⁵ pooled splenocytes (means + SD). Data are from one representative experiment of two performed.