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

Calvet CY, Thalmensi J, Liard C, Pliquet E, Bestetti T, Huet T, Langlade-Demoyen P, Mir LM - Mol Ther Methods Clin Dev (2014)

Bottom Line: Gene electrotransfer is the most efficient and safest non-viral gene transfer procedure and specific electrical parameters have been developed for several target tissues.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.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris-Sud, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses , Villejuif, France ; CNRS, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses , Villejuif, France ; Gustave Roussy, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses , Villejuif, France.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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fig6: 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.

Mentions: We performed an evaluation of the cytolytic strength of the hTERT-specific CD8 T-cells using carboxyfluorescein succinimidyl ester (CFSE)-labeled and peptide-pulsed splenocytes as target cells, as mentioned in the Materials and Methods section. Indeed, the cytotoxic activity of CTL is mandatory to kill tumor cells. In order to finally validate the EGT Pd parameters that could be used in clinical trials, we compared the cytotoxic activity of hTERT-specific CD8 T-cells in C57BL/6J mice immunized through the ID route using INVAC-1 combined with two distinct EGT parameters: one HV pulse of 700 V/cm and of 100 µs followed 1,000 ms later by one LV pulse of 100 V/cm and of 400 ms (optimal parameters for EGT in muscle,1 further referred as Pm) or Pd parameters. In the context of a single immunization with INVAC-1 plasmid, we showed that EGT performed with Pd was associated with a twofold increase in the lysis of target cells bearing class I MHC-restricted telomerase peptides (50% of specific lysis), as compared to EGT performed with Pm (25% of specific lysis) (Figure 6), even though this difference was not statistically significant due to high standard deviations. Thus, INVAC-1–mediated ID vaccination using efficient EGT parameters can generate hTERT-specific CTL.


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

Calvet CY, Thalmensi J, Liard C, Pliquet E, Bestetti T, Huet T, Langlade-Demoyen P, Mir LM - Mol Ther Methods Clin Dev (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362362&req=5

fig6: 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.
Mentions: We performed an evaluation of the cytolytic strength of the hTERT-specific CD8 T-cells using carboxyfluorescein succinimidyl ester (CFSE)-labeled and peptide-pulsed splenocytes as target cells, as mentioned in the Materials and Methods section. Indeed, the cytotoxic activity of CTL is mandatory to kill tumor cells. In order to finally validate the EGT Pd parameters that could be used in clinical trials, we compared the cytotoxic activity of hTERT-specific CD8 T-cells in C57BL/6J mice immunized through the ID route using INVAC-1 combined with two distinct EGT parameters: one HV pulse of 700 V/cm and of 100 µs followed 1,000 ms later by one LV pulse of 100 V/cm and of 400 ms (optimal parameters for EGT in muscle,1 further referred as Pm) or Pd parameters. In the context of a single immunization with INVAC-1 plasmid, we showed that EGT performed with Pd was associated with a twofold increase in the lysis of target cells bearing class I MHC-restricted telomerase peptides (50% of specific lysis), as compared to EGT performed with Pm (25% of specific lysis) (Figure 6), even though this difference was not statistically significant due to high standard deviations. Thus, INVAC-1–mediated ID vaccination using efficient EGT parameters can generate hTERT-specific CTL.

Bottom Line: Gene electrotransfer is the most efficient and safest non-viral gene transfer procedure and specific electrical parameters have been developed for several target tissues.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.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris-Sud, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses , Villejuif, France ; CNRS, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses , Villejuif, France ; Gustave Roussy, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses , Villejuif, France.

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.

No MeSH data available.


Related in: MedlinePlus