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Targeted Collection of Plasmid DNA in Large and Growing Animal Muscles 6 Weeks after DNA Vaccination with and without Electroporation.

Dory D, Le Moigne V, Cariolet R, Béven V, Keranflec'h A, Jestin A - J Immunol Res (2015)

Bottom Line: Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy.This is even more difficult for large and growing animals.Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

View Article: PubMed Central - PubMed

Affiliation: French Agency for Food, Environmental and Occupational Health Safety (ANSES), Viral Genetics and Biosafety Unit, 22440 Ploufragan, France.

ABSTRACT
DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

No MeSH data available.


Related in: MedlinePlus

Plasmid concentrations in the different muscle fractions six weeks after injection. Two groups of four pigs were intramuscularly injected with 2.5 × 1014 copies of PrV-gB-pcDNA3 with or without electroporation, respectively. Six weeks later, excision of the muscle injection site was performed using a disposable 2 cm long and 0.8 cm diameter biopsy punch. (a) The excised muscle portion was divided into six fractions, F1 to F6. Fraction F1 represents the most external part of the muscle (i.e., under the skin) and F6 the most internal part. Thereafter plasmid DNA concentration was measured in each fraction after DNA extraction. Levels (in number of plasmid copies per μg of total DNA) of PrV-gB-pcDNA3 present in injected or opposite (noninjected) biceps femoris were quantified by real-time qPCR. Individual plasmid concentrations in each muscle sample for each pig injected with (b) or without (c) electroporation are presented. When taking into account the mean values for each fraction (not shown), the differences between both groups were not statistically significant (P > 0.05, nonparametric Mann-Whitney test).
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fig3: Plasmid concentrations in the different muscle fractions six weeks after injection. Two groups of four pigs were intramuscularly injected with 2.5 × 1014 copies of PrV-gB-pcDNA3 with or without electroporation, respectively. Six weeks later, excision of the muscle injection site was performed using a disposable 2 cm long and 0.8 cm diameter biopsy punch. (a) The excised muscle portion was divided into six fractions, F1 to F6. Fraction F1 represents the most external part of the muscle (i.e., under the skin) and F6 the most internal part. Thereafter plasmid DNA concentration was measured in each fraction after DNA extraction. Levels (in number of plasmid copies per μg of total DNA) of PrV-gB-pcDNA3 present in injected or opposite (noninjected) biceps femoris were quantified by real-time qPCR. Individual plasmid concentrations in each muscle sample for each pig injected with (b) or without (c) electroporation are presented. When taking into account the mean values for each fraction (not shown), the differences between both groups were not statistically significant (P > 0.05, nonparametric Mann-Whitney test).

Mentions: Four points were tattooed with Indian ink on the skin of the left biceps femoris muscle of 5-week-old piglets (as shown in Figure 1). When the piglets were eight-week old, they were i.m. injected either with PrV-gB-pcDNA3 followed or not by electroporation, with pcDNA3 and electroporation, or with PBS and electroporation. The injection site was located at the intersection of the two lines passing through these dots (Figure 1). The mean weight of the animals at the vaccination time was of 24.7 ± 2.1 kg. No adverse reactions, no fever, and no delay in the growth of the pigs were observed during the 6-week period following these injections. The mean weight of the animals 6 weeks after injection was 66.5 ± 5.4 kg. This means that the piglets gained 41.8 ± 4.0 kg during the whole experimental period (i.e., about 170% of their initial weight). In a previous report we showed that plasmid injection coupled with electroporation applied exactly in the same manner as here increased the production of specific antibodies against PrV and peripheral blood mononuclear cells proliferated in response to stimulation with PrV glycoproteins [9]. This means that the electroporation conditions used here are effective ones. At week 6 after injection, 2 cm long muscle samples were collected exactly at the injection site using the dots tattooed on the skin (Figure 1). This time-point was the same as used in the study describing the research of integration events by RAIC-PCR [16]. Furthermore, this time-point seemed to us realistic since we detected small amounts of plasmids in the injected muscle 21 days after DNA vaccination, without electroporation, with much less plasmids injected, and without identifying precisely the injection point as done here [14]. At this time-point, the four tattooed dots were still strongly marked. The two lines delineating the injection site were drawn again on the skin (as shown in Figure 1). After removing the skin and the fat layer, the portion of the injected muscle was sampled using a disposable 2 cm long and 0.8 cm diameter biopsy punch that was horizontally applied on the muscle surface (Figure 2), frozen in liquid nitrogen, and stored at −80°C until DNA extraction. In preliminary experiments, the injection point was found to be located approximately in the middle of the muscle sample (data not shown). No plasmids were detected in pigs injected with PBS. The fractions containing the highest concentration of plasmids (around 3,000 to 14,000 copies/μg of host DNA) are located around the middle of the muscle samples, between fractions F2 and F5, with 6 out of 8 pigs within fractions F3 or F4 (Figure 3). The concentrations were higher in F2–F4 (1 pig in F2, 2 pigs in F3, and 1 pig in F4) when electroporation was applied and in F4-F5 (3 pigs in F4 and 1 pig in F5), that is, deeper, in the other case. When taking into account the mean values for each fraction, the concentrations of plasmids within the electropored muscle fractions were not significantly higher than in the nonelectropored ones (P > 0.05) (not shown). But these observations have to be taken with caution since the pressure we applied to the device was not controlled, although we tried. Nevertheless this seems consistent with the fact that less diffusion of the plasmids and better precision of injection are observed with electroporation [21]. Importantly, the fraction which is at the end of the needle is restricted to a small area, at least in depth, which shows the usefulness of precise benchmarks. Finally all the electropored and nonelectropored muscle samples have a concentration of plasmid DNA inferior to 30,000 copies/μg of host DNA. If we take into account the recommendations of the FDA [22], it is not necessary to perform additional integration analyses of plasmid DNA into host DNA since the probability of integration is low (<30,000 copies of plasmid/μg of host DNA).


Targeted Collection of Plasmid DNA in Large and Growing Animal Muscles 6 Weeks after DNA Vaccination with and without Electroporation.

Dory D, Le Moigne V, Cariolet R, Béven V, Keranflec'h A, Jestin A - J Immunol Res (2015)

Plasmid concentrations in the different muscle fractions six weeks after injection. Two groups of four pigs were intramuscularly injected with 2.5 × 1014 copies of PrV-gB-pcDNA3 with or without electroporation, respectively. Six weeks later, excision of the muscle injection site was performed using a disposable 2 cm long and 0.8 cm diameter biopsy punch. (a) The excised muscle portion was divided into six fractions, F1 to F6. Fraction F1 represents the most external part of the muscle (i.e., under the skin) and F6 the most internal part. Thereafter plasmid DNA concentration was measured in each fraction after DNA extraction. Levels (in number of plasmid copies per μg of total DNA) of PrV-gB-pcDNA3 present in injected or opposite (noninjected) biceps femoris were quantified by real-time qPCR. Individual plasmid concentrations in each muscle sample for each pig injected with (b) or without (c) electroporation are presented. When taking into account the mean values for each fraction (not shown), the differences between both groups were not statistically significant (P > 0.05, nonparametric Mann-Whitney test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Plasmid concentrations in the different muscle fractions six weeks after injection. Two groups of four pigs were intramuscularly injected with 2.5 × 1014 copies of PrV-gB-pcDNA3 with or without electroporation, respectively. Six weeks later, excision of the muscle injection site was performed using a disposable 2 cm long and 0.8 cm diameter biopsy punch. (a) The excised muscle portion was divided into six fractions, F1 to F6. Fraction F1 represents the most external part of the muscle (i.e., under the skin) and F6 the most internal part. Thereafter plasmid DNA concentration was measured in each fraction after DNA extraction. Levels (in number of plasmid copies per μg of total DNA) of PrV-gB-pcDNA3 present in injected or opposite (noninjected) biceps femoris were quantified by real-time qPCR. Individual plasmid concentrations in each muscle sample for each pig injected with (b) or without (c) electroporation are presented. When taking into account the mean values for each fraction (not shown), the differences between both groups were not statistically significant (P > 0.05, nonparametric Mann-Whitney test).
Mentions: Four points were tattooed with Indian ink on the skin of the left biceps femoris muscle of 5-week-old piglets (as shown in Figure 1). When the piglets were eight-week old, they were i.m. injected either with PrV-gB-pcDNA3 followed or not by electroporation, with pcDNA3 and electroporation, or with PBS and electroporation. The injection site was located at the intersection of the two lines passing through these dots (Figure 1). The mean weight of the animals at the vaccination time was of 24.7 ± 2.1 kg. No adverse reactions, no fever, and no delay in the growth of the pigs were observed during the 6-week period following these injections. The mean weight of the animals 6 weeks after injection was 66.5 ± 5.4 kg. This means that the piglets gained 41.8 ± 4.0 kg during the whole experimental period (i.e., about 170% of their initial weight). In a previous report we showed that plasmid injection coupled with electroporation applied exactly in the same manner as here increased the production of specific antibodies against PrV and peripheral blood mononuclear cells proliferated in response to stimulation with PrV glycoproteins [9]. This means that the electroporation conditions used here are effective ones. At week 6 after injection, 2 cm long muscle samples were collected exactly at the injection site using the dots tattooed on the skin (Figure 1). This time-point was the same as used in the study describing the research of integration events by RAIC-PCR [16]. Furthermore, this time-point seemed to us realistic since we detected small amounts of plasmids in the injected muscle 21 days after DNA vaccination, without electroporation, with much less plasmids injected, and without identifying precisely the injection point as done here [14]. At this time-point, the four tattooed dots were still strongly marked. The two lines delineating the injection site were drawn again on the skin (as shown in Figure 1). After removing the skin and the fat layer, the portion of the injected muscle was sampled using a disposable 2 cm long and 0.8 cm diameter biopsy punch that was horizontally applied on the muscle surface (Figure 2), frozen in liquid nitrogen, and stored at −80°C until DNA extraction. In preliminary experiments, the injection point was found to be located approximately in the middle of the muscle sample (data not shown). No plasmids were detected in pigs injected with PBS. The fractions containing the highest concentration of plasmids (around 3,000 to 14,000 copies/μg of host DNA) are located around the middle of the muscle samples, between fractions F2 and F5, with 6 out of 8 pigs within fractions F3 or F4 (Figure 3). The concentrations were higher in F2–F4 (1 pig in F2, 2 pigs in F3, and 1 pig in F4) when electroporation was applied and in F4-F5 (3 pigs in F4 and 1 pig in F5), that is, deeper, in the other case. When taking into account the mean values for each fraction, the concentrations of plasmids within the electropored muscle fractions were not significantly higher than in the nonelectropored ones (P > 0.05) (not shown). But these observations have to be taken with caution since the pressure we applied to the device was not controlled, although we tried. Nevertheless this seems consistent with the fact that less diffusion of the plasmids and better precision of injection are observed with electroporation [21]. Importantly, the fraction which is at the end of the needle is restricted to a small area, at least in depth, which shows the usefulness of precise benchmarks. Finally all the electropored and nonelectropored muscle samples have a concentration of plasmid DNA inferior to 30,000 copies/μg of host DNA. If we take into account the recommendations of the FDA [22], it is not necessary to perform additional integration analyses of plasmid DNA into host DNA since the probability of integration is low (<30,000 copies of plasmid/μg of host DNA).

Bottom Line: Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy.This is even more difficult for large and growing animals.Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

View Article: PubMed Central - PubMed

Affiliation: French Agency for Food, Environmental and Occupational Health Safety (ANSES), Viral Genetics and Biosafety Unit, 22440 Ploufragan, France.

ABSTRACT
DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

No MeSH data available.


Related in: MedlinePlus