Optimization of a gene electrotransfer procedure for efficient intradermal immunization with an hTERT-based DNA vaccine in mice

Abstract

DNA vaccination consists in administering an antigen-encoding plasmid in order to trigger a specific immune response. This specific vaccine strategy is of particular interest to fight against various infectious diseases and cancer. Gene electrotransfer is the most efficient and safest non-viral gene transfer procedure and specific electrical parameters have been developed for several target tissues. Here, a gene electrotransfer protocol into the skin has been optimized in mice for efficient intradermal immunization against the well-known telomerase tumor antigen. First, the luciferase reporter gene was used to evaluate gene electrotransfer efficiency into the skin as a function of the electrical parameters and electrodes, either non-invasive or invasive. In a second time, these parameters were tested for their potency to generate specific cellular CD8 immune responses against telomerase epitopes. These CD8 T-cells were fully functional as they secreted IFNγ and were endowed with specific cytotoxic activity towards target cells. This simple and optimized procedure for efficient gene electrotransfer into the skin using the telomerase antigen is to be used in cancer patients for the phase 1 clinical evaluation of a therapeutic cancer DNA vaccine called INVAC-1.

Figure 1

Comparison of pCMV-luc gene transfer into the dermis and INVAC-1-mediated ID vaccination efficiencies with or without EP using plate electrodes. (a) Representation of bioluminescence intensities in C57BL/6J mice 2 days after pCMV-luc ID injection followed or not by EP, n = 5 mice for pCMV-luc ID injection alone, n = 10 (from 5 mice, 2 treatments per mouse) for pCMV-luc ID injection+EP. (b) Frequency of hTERT-specific IFNγ⁺ CD8 T-cells detected in C57BL/6J mice vaccinated 14 days before with 25 µg of INVAC-1 followed or not by EP, n = 8 both for INVAC-1 ID injection alone or n = 6 for INVAC-1 ID injection+EP. Bars represent median values, *P < 0.05, **P < 0.01, Mann–Whitney–Wilcoxon test.

Figure 2

Choice of the best electrodes for pCMV-luc electrotransfer into the dermis and INVAC-1–mediated ID vaccination. (a) Representation of bioluminescence intensities 2 days after pCMV-luc electrotransfer in C57BL/6J mice using the three types of electrodes, n = 14 mice for pCMV-luc ID injection alone, n = 8–10 (from four to five mice, two treatments per mouse) for pCMV-luc ID injection+EP. (b) Frequency of hTERT-specific INFγ⁺ CD8 T-cells detected in HLA-B7 mice vaccinated intradermally 14 days before with INVAC-1 using the three types of electrodes, n = 3 mice for PBS immunization control and n = 4–9 mice for INVAC-1–mediated immunization. Bars represent median values, *P < 0.05, ***P < 0.001, Kruskal–Wallis test with Dunn’s multiple comparison test.

Figure 3

Localization of luciferase expression after ID injection and electrotransfer of pCMV-luc into the dermal layer of C57BL/6J mice using plate electrodes. Bioluminescence intensities were evaluated in the skin flap and in the underlying muscles 4 days after ID injection of pCMV-luc and EGT, n = 3 mice.

Figure 4

Determination of the optimal HV pulse intensity for intradermally injected pCMV-luc electrotransfer in C57BL/6J mice using plate electrodes. Bioluminescence intensities were evaluated 2 days after pCMV-luc electrotransfer performed in C57BL/6J mice. One single HV pulse of different intensities in combination with one LV pulse of 140 V/cm were compared, n = 24 mice for pCMV-luc ID injection alone, n = 8 (from 4 mice, 2 treatments per mouse) for pCMV-luc ID injection+EP. Bars represent median values, *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal–Wallis test with Dunn’s multiple comparison test.

Figure 5

Choice of the best HV-LV pulses combination for pCMV-luc dermal electrotransfer and INVAC-1–mediated ID vaccination. (a) Bioluminescence obtained in C57BL/6J mice 2 days after pCMV-luc ID injection upon various HV-LV pulses combinations, n = 30 mice for pCMV-luc ID injection alone and n = 6 (from 3 mice, 2 treatments per mouse) for pCMV-luc ID injection+EP delivered onto the skin. (b) Frequency of hTERT-specific IFNγ⁺ CD8 T-cells detected in C57BL/6J mice vaccinated 14 days before with INVAC-1 according to various combinations of HV-LV pulses, n = 8 mice for PBS immunization control and n = 5 mice for INVAC-1–mediated immunization. (c) Comparison of the bioluminescence intensities in C57BL/6J mice in which EGT was performed 2 days before with P9 or Pd, n = 4 mice for pCMV-luc ID injection alone, n = 8 (from four mice, two treatments per mouse) for pCMV-luc ID injection+EP. (d) Comparison of the frequency of hTERT-specific IFNγ⁺ CD8 T-cells in C57BL/6J mice immunized intradermally 14 days before by using INVAC-1 combined with P9 or Pd, n = 4 mice for PBS immunization control and n = 6 mice for INVAC-1–mediated immunization. Bars represent median values. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal–Wallis test with Dunn’s multiple comparison test.

Figure 6

Evaluation of the hTERT-specific cytotoxicity of CD8 T-cells in immunized mice as a function of the dermal electrotransfer parameters used. C57BL/6J mice were intradermally immunized with INVAC-1 using Pd or Pm parameters. 14 days later, splenocytes from naïve mice were recovered, stained with CFSE, pulsed with p660 peptides and eventually injected i.v. in immunized mice. 15 hours later, spleens from immunized mice were recovered and CFSE-labeled splenocytes were quantified by flow cytometry, n = 8 mice. Bars represent median values, ns for not statistically significant, Mann–Whitney–Wilcoxon test.

Figure 7

INVAC-1 plasmid map. Bases 1–3478: NTC8685-eRNA41H-HindIII-XbaI vector (NTC); Bases 3479–3484: HindIII cloning site (NTC/Invectys); Bases 3485–6967: Ubi-Telomerase transgene (Invectys); Bases 6968–6973: XbaI cloning site (Invectys/NTC); Bases 6974–7120: NTC8685-eRNA41H-HindIII-XbaI vector (NTC).

Figure 8

Electrodes used in this study. (a) Non-invasive plate electrodes. (b) Invasive needle electrodes. (c) Invasive finger electrodes.