Abstract
BackgroundApplication 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.MethodsA 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 – 109 copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.ResultsPersistence 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.ConclusionQuantitative 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.
Highlights
Application of plasmid DNA for immunization of food-producing animals established new standards of food safety
The Quantitative real-time PCR (QRTPCR) method was used for the study of the persistence of plasmid DNA (pDNA) at the injection sites in mice and beef cattle. For this reason we developed an isolation and detection QRTPCR based methodology for the accurate quantification of residual levels of vaccine pDNAX in the muscles after various approaches to vaccine application
Validation of QRTPCR method Persistence of pDNAX was determined by a QRTPCR methodology designed to recognize the stretch of promoter-insert from the pDNAX plasmid
Summary
Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. DNA-based vaccines represent a new and rapidly progressing area in vaccinology. Plasmid DNA (pDNA) vaccines have been reported to induce protective immunity in numerous animal models of parasitic, viral and bacterial diseases [1]. Persistent plasmid does not replicate inside the cells [7] and there are no significant increases in anti-DNA antibodies leading to autoimmune reactions [9]. Preclinical studies on animal models document overall safety, some issues and potential risks related to food-producing animals need to be addressed directly on target species since these represent separate issues to clinical applications. Data on the rates of clearance, or persistence, of pDNA post injection into animals is only limited, potential risks must be extrapolated from model animal studies. All the studies have given evidence for overall safety as well
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