Limits...
Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles.

Orság P, Kvardová V, Raska M, Miller AD, Ledvina M, Turánek J - Genet Vaccines Ther (2008)

Bottom Line: The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.The linear quantification range and the sensitivity of the method was found to be 10 - 10(9) copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.Residual plasmid in native state will hardly be found at measurable level following further meat processing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Veterinary Research Institute, Department of Immunology, Brno, Czech Republic. petr.orsag@gmail.com

ABSTRACT

Background: Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.

Methods: A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 - 10(9) copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.

Results: Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242-292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.

Conclusion: Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.

No MeSH data available.


Related in: MedlinePlus

Levels of pDNAX detected by QRTPCR in calf muscle (at the injection site) after administration of 10 μg pDNAX in 5-week old BalB/C mice. The line connects the average levels of plasmid DNA expressed in logarithm scale detected by QRTPCR in 500 ng of isolated DNA (MC/r) ± SD (four mice per time point). The straight line represent quantification limit of QRTPCR assay (10 pDNAX copies/reaction). The dotted line represent detection limit of QRTPCR assay (3 pDNAX copies/reaction). The data from control group were omitted (all control animals were negative). Routes of application: full circle denotes naked pDNAX; full triangle denotes pDNAX plus electroporation; full square denotes pDNAX:CDAN-DOPE complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2542361&req=5

Figure 3: Levels of pDNAX detected by QRTPCR in calf muscle (at the injection site) after administration of 10 μg pDNAX in 5-week old BalB/C mice. The line connects the average levels of plasmid DNA expressed in logarithm scale detected by QRTPCR in 500 ng of isolated DNA (MC/r) ± SD (four mice per time point). The straight line represent quantification limit of QRTPCR assay (10 pDNAX copies/reaction). The dotted line represent detection limit of QRTPCR assay (3 pDNAX copies/reaction). The data from control group were omitted (all control animals were negative). Routes of application: full circle denotes naked pDNAX; full triangle denotes pDNAX plus electroporation; full square denotes pDNAX:CDAN-DOPE complex.

Mentions: Identical experiment was performed with 5-week-old mice to evaluate a possible relationship between the animal age and the rate of clearance of pDNAX from the site of injection. In both cases, naked pDNAX and pDNAX electroporation groups, the rates of clearance of pDNAX were found to be slower for 5-week-old mice in comparison to the corresponding situation in 8-week-old mice (compare Fig. 2 and Fig. 3). Nevertheless, the final differences in pDNAX levels between pDNAX:CDAN/DOPE and the pDNAX electroporation groups were still in the range of 100-fold, with an even greater gap of over 104-fold between pDNAX:CDAN/DOPE and naked pDNAX groups. In this instance too, a difference of 1–2 orders of magnitude also existed between the measured plasmid levels in the pDNAX electroporation group and the naked pDNAX group at all time points analyzed (Fig. 3), in partial contrast to our observations with 8-week animals (Fig. 2).


Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles.

Orság P, Kvardová V, Raska M, Miller AD, Ledvina M, Turánek J - Genet Vaccines Ther (2008)

Levels of pDNAX detected by QRTPCR in calf muscle (at the injection site) after administration of 10 μg pDNAX in 5-week old BalB/C mice. The line connects the average levels of plasmid DNA expressed in logarithm scale detected by QRTPCR in 500 ng of isolated DNA (MC/r) ± SD (four mice per time point). The straight line represent quantification limit of QRTPCR assay (10 pDNAX copies/reaction). The dotted line represent detection limit of QRTPCR assay (3 pDNAX copies/reaction). The data from control group were omitted (all control animals were negative). Routes of application: full circle denotes naked pDNAX; full triangle denotes pDNAX plus electroporation; full square denotes pDNAX:CDAN-DOPE complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2542361&req=5

Figure 3: Levels of pDNAX detected by QRTPCR in calf muscle (at the injection site) after administration of 10 μg pDNAX in 5-week old BalB/C mice. The line connects the average levels of plasmid DNA expressed in logarithm scale detected by QRTPCR in 500 ng of isolated DNA (MC/r) ± SD (four mice per time point). The straight line represent quantification limit of QRTPCR assay (10 pDNAX copies/reaction). The dotted line represent detection limit of QRTPCR assay (3 pDNAX copies/reaction). The data from control group were omitted (all control animals were negative). Routes of application: full circle denotes naked pDNAX; full triangle denotes pDNAX plus electroporation; full square denotes pDNAX:CDAN-DOPE complex.
Mentions: Identical experiment was performed with 5-week-old mice to evaluate a possible relationship between the animal age and the rate of clearance of pDNAX from the site of injection. In both cases, naked pDNAX and pDNAX electroporation groups, the rates of clearance of pDNAX were found to be slower for 5-week-old mice in comparison to the corresponding situation in 8-week-old mice (compare Fig. 2 and Fig. 3). Nevertheless, the final differences in pDNAX levels between pDNAX:CDAN/DOPE and the pDNAX electroporation groups were still in the range of 100-fold, with an even greater gap of over 104-fold between pDNAX:CDAN/DOPE and naked pDNAX groups. In this instance too, a difference of 1–2 orders of magnitude also existed between the measured plasmid levels in the pDNAX electroporation group and the naked pDNAX group at all time points analyzed (Fig. 3), in partial contrast to our observations with 8-week animals (Fig. 2).

Bottom Line: The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.The linear quantification range and the sensitivity of the method was found to be 10 - 10(9) copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.Residual plasmid in native state will hardly be found at measurable level following further meat processing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Veterinary Research Institute, Department of Immunology, Brno, Czech Republic. petr.orsag@gmail.com

ABSTRACT

Background: Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.

Methods: A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 - 10(9) copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.

Results: Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242-292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.

Conclusion: Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.

No MeSH data available.


Related in: MedlinePlus