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Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporation.

Mendoza JM, Amante DH, Kichaev G, Knott CL, Kiosses WB, Smith TR, Sardesai NY, Broderick KE - Vaccines (Basel) (2013)

Bottom Line: Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen.In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space.These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

View Article: PubMed Central - PubMed

Affiliation: Inovio Pharmaceuticals Inc., 1787 Sentry Parkway West, Building 18, Suite 400, Blue Bell, PA 19422, USA.

ABSTRACT
The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP) device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21) of enhanced gene delivery with electroporation (EP) was performed to observe the localization of green fluorescent protein (GFP) expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

No MeSH data available.


Histological analysis reveals infiltration of lymphocytes at the EP treatment site. Guinea pig skin sections were immunohistochemically stained with an antibody recognizing lymphocytes and Langerhans cells (Tetramethylrhodamine isothiocyanate (TRITC) secondary) after ID GFP plasmid administration followed by EP with SEP. Skin biopsies were removed, cryosectioned, antibody and 4',6-diamidino-2-phenylindole (DAPI) stained and visualized under fluorescence microscopy at 20× and 40×. An untreated control (no DNA, no EP) is also shown.
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vaccines-01-00384-f005: Histological analysis reveals infiltration of lymphocytes at the EP treatment site. Guinea pig skin sections were immunohistochemically stained with an antibody recognizing lymphocytes and Langerhans cells (Tetramethylrhodamine isothiocyanate (TRITC) secondary) after ID GFP plasmid administration followed by EP with SEP. Skin biopsies were removed, cryosectioned, antibody and 4',6-diamidino-2-phenylindole (DAPI) stained and visualized under fluorescence microscopy at 20× and 40×. An untreated control (no DNA, no EP) is also shown.

Mentions: The major APC in the epidermis is the Langerhans cell. Using an antibody against Langerhans cells and lymphocytes, we immunostained skin sections to observe the dynamics of these migratory cells following EP. Although increased numbers of lymphocytic cells were observed following DNA injection alone and EP alone (data not shown), a significant increase in cell numbers was observed following EP enhanced delivery of plasmid expressing GFP at 6 h post treatment (Figure 5). Through analysis of multiple GFP treated sections, lymphocytic cells could be seen migrating towards and congregating in the areas of the skin biopsy where the transfected GFP expressing cells appeared.


Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporation.

Mendoza JM, Amante DH, Kichaev G, Knott CL, Kiosses WB, Smith TR, Sardesai NY, Broderick KE - Vaccines (Basel) (2013)

Histological analysis reveals infiltration of lymphocytes at the EP treatment site. Guinea pig skin sections were immunohistochemically stained with an antibody recognizing lymphocytes and Langerhans cells (Tetramethylrhodamine isothiocyanate (TRITC) secondary) after ID GFP plasmid administration followed by EP with SEP. Skin biopsies were removed, cryosectioned, antibody and 4',6-diamidino-2-phenylindole (DAPI) stained and visualized under fluorescence microscopy at 20× and 40×. An untreated control (no DNA, no EP) is also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494224&req=5

vaccines-01-00384-f005: Histological analysis reveals infiltration of lymphocytes at the EP treatment site. Guinea pig skin sections were immunohistochemically stained with an antibody recognizing lymphocytes and Langerhans cells (Tetramethylrhodamine isothiocyanate (TRITC) secondary) after ID GFP plasmid administration followed by EP with SEP. Skin biopsies were removed, cryosectioned, antibody and 4',6-diamidino-2-phenylindole (DAPI) stained and visualized under fluorescence microscopy at 20× and 40×. An untreated control (no DNA, no EP) is also shown.
Mentions: The major APC in the epidermis is the Langerhans cell. Using an antibody against Langerhans cells and lymphocytes, we immunostained skin sections to observe the dynamics of these migratory cells following EP. Although increased numbers of lymphocytic cells were observed following DNA injection alone and EP alone (data not shown), a significant increase in cell numbers was observed following EP enhanced delivery of plasmid expressing GFP at 6 h post treatment (Figure 5). Through analysis of multiple GFP treated sections, lymphocytic cells could be seen migrating towards and congregating in the areas of the skin biopsy where the transfected GFP expressing cells appeared.

Bottom Line: Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen.In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space.These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

View Article: PubMed Central - PubMed

Affiliation: Inovio Pharmaceuticals Inc., 1787 Sentry Parkway West, Building 18, Suite 400, Blue Bell, PA 19422, USA.

ABSTRACT
The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP) device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21) of enhanced gene delivery with electroporation (EP) was performed to observe the localization of green fluorescent protein (GFP) expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

No MeSH data available.