Enhancement of DNA vaccine potency through coadministration of CIITA DNA with DNA vaccines via gene gun

Abstract

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.

Figure 1. Flow cytometry analysis to demonstrate the expression of murine MHC molecules in CIITA-transfected DC-1 cells

Flow cytometry data demonstrating the levels of H-2 K^b, H-2 D^b, and I-A^b expression in CIITA-transfected cells and controls. The legend shows the vector plasmids with which the DCs were transfected. An immortalized dendritic cell line (DC-1) was transfected with CIITA or mutant CIITA (mtCIITA). Untransfected cells and cells transfected with the pcDNA3 vector backbone were used as a control. The expression of MHC I and II molecules was characterized using antibodies to MHC I H-2 K^b, H-2 D^b, and MHC II I-A^b by flow cytometry analysis.

Figure 2. Characterization of the MHC class I and II presentation of DCs transfected with CIITA DNA

DCs were cotransfected with CIITA DNA and CRT/E6 DNA (A & B) or Ii-PADRE DNA (C & D). The DCs were then incubated with E6-specific CD8+ T cells (A & B) or PADRE-specific CD4+ T cells (C & D) overnight. The activation of antigen-specific T cells was characterized by intracellular cytokine staining followed by flow cytometry analysis using IFN-γ and CD4 or CD8-specific antibodies. A and C. Representative flow cytometry data showing the numbers of activated E6-specific CD8+ T cells (A) and PADRE-specific CD4⁺ T cells (C) after incubation with the cotransfected DCs. B and D. Bar graphs depicting the numbers of E6-specific CD8+ T cells (B) and PADRE-specific CD4⁺ T cells (D) (means±s.d.). The data presented in this figure are from one representative experiment of two performed.

Figure 3. Characterization of the E6-specific CD8+ T cell immune responses in mice vaccinated with CIITA DNA and CRT/E6 DNA

C57BL/6 mice (5 per group) were immunized with 2 μg /mouse of CIITA and/or CRT/E6 DNA twice with a 1-week interval. Splenocytes from vaccinated mice were harvested 1 week after the last vaccination and characterized for E6-specific CD8⁺ T cells using intracellular IFN-γ staining followed by flow cytometry analysis. A. Representative flow cytometry data for the E6-specific CD8+ T cell immune responses. The numbers in the upper right-hand corner represent the number of E6-specific IFN-γ-secreting CD8+ T cells per 5×10⁶ pooled splenocytes. B. Bar graphs depicting the numbers of E6-specific IFN-γ-secreting CD8⁺ T cells per 5×10⁶ pooled splenocytes (means±s.d.). The data presented in this figure are from one representative experiment of two performed.

Figure 4. Characterization of the E6-specific CD8⁺ T cells in mice vaccinated with CRT/E6, CIITA DNA and Ii-PADRE DNA vaccines

C57BL/6 mice (5 per group) were immunized with 2 μg /mouse twice with a 1-wk interval of the DNA combinations listed in Table 1. Splenocytes from vaccinated mice were harvested 1 week after the last vaccination and characterized for E6-specific CD8⁺ T cells using intracellular IFN-γ staining followed by flow cytometry analysis. A. Representative flow cytometry data. The numbers in the upper right-hand corner represent the number of E6-specific IFN-γ-secreting CD8⁺ T cells per 5×10⁶ pooled splenocytes. B. Bar graphs depicting the numbers of E6-specific IFN-γ-secreting CD8⁺ T cells per 5×10⁶ pooled splenocytes (means±s.d.). The data presented in this figure are from one representative experiment of two performed.

Figure 5. In vivo tumor treatment experiments

C57BL/6 mice (5 per group) were first challenged with 5×10⁴/mouse of TC-1 tumor cells by subcutaneous injection. Three days after tumor challenge, the mice were administered 2 μg DNA/mouse 3 times with 4-day intervals of the various DNA vaccine mixtures listed in Table 1. The mice were monitored for evidence of tumor growth by inspection and palpation twice a week. Tumor volumes were measured starting from day 7 after tumor challenge. (A) Line graph depicting the tumor volumes in mice of different tumor treatments (means±s.d.). (B) Kaplan & Meier survival analysis in mice of the tumor treatment experiments. The data shown here are from one representative experiment of two performed.

Figure 6. Characterization of the long-term E6-specific CD8⁺ T cell immune response in mice vaccinated with CRT/E6, CIITA DNA and Ii-PADRE DNA vaccines

C57BL/6 mice (5 per group) were immunized with 2 μg /mouse twice with a 1-wk interval of the DNA combinations listed in Table 1. Splenocytes from vaccinated mice were harvested 60 days after the last vaccination and characterized for E6-specific CD8⁺ T cells using intracellular IFN-γ staining followed by flow cytometry analysis. A. Representative flow cytometry data. The numbers in the upper right-hand corner represent the number of memory E6-specific IFN-γ-secreting CD8⁺ T per 5×10⁶ pooled splenocytes. B. Bar graphs depicting the numbers of memory E6-specific IFN-γ-secreting CD8⁺ T cells per 5×10⁶ pooled splenocytes (means±s.d.). The data presented in this figure are from one representative experiment of two performed.

Figure 7. Long-term in vivo tumor protection experiments

C57BL/6 mice (5 per group) were immunized with 2 μg DNA/mouse twice with a 1-week interval of the various DNA vaccine mixtures listed in Table 1. Two months after the last vaccination, the mice were challenged by subcutaneous injection of 1×10⁵/mouse of TC-1 cells. The mice were monitored for evidence of tumor growth by inspection and palpation twice a week. Tumor volumes were measured starting from day 7 after tumor challenge. (A) Line graph depicting tumor volume in mice challenged with TC-1 cells (means±s.d.). (B) Kaplan & Meier survival analysis in mice challenged with TC-1 cells. The data shown here are from one representative experiment of two performed.