Biodistribution Of Avian Influenza DNA Vaccine In Chicken

HPAI is considered a kind of strong infectious diseases by OIE and a infectious disease of animal by BWC.It has many serological types and strong variability, at present,vaccine is the most efficient measure to prevent it. As a kind of new genetically engineering vaccine,DNA vaccine has many preponderances than tradional vaccines, such as induce general immunological response,immunity persistence,it is easy to composite combined vaccine, multivalent vaccine or engomphosis vaccine.However,we must think highly of its risks,most important is integration risk.10μg of H5 subtype avian influenza DNA vaccine pCAGGoptiHA5 could induce complete protection in chickens and persisted one year.In order to reveal the mechanism of plasmid DNA vaccine permanent immunity, we used quantitative real-time PCR to define the distribution and persistence of DNA after immunity.An integrati-on study was conducted in chickens to determine whether pDNA integration into the genome of the vaccinated animal.According to the conserved region of pCAGGoptiHA5,we desigened primers and TaqMan MGB probes,then choose DNA vaccine pCAGGoptiHA5 as standardize to establish SYBR Green I quantitative real-time PCR (QPCR) and TaqMan MGB QPCR methods.After compared specificity,sensibility,reproducibility and contamination,we found that the correlation is above 0.999 and the sensibility is 42copies,but the reproducibility and contamination of TaqMan MGB QPCR are better than SYBR Green I QPCR.We choose TaqMan MGB QPCR to study the biodistribution and persistence of plasmid DNA vaccine pCAGGoptiHA5 in chickens.We divided chickens into 3 groups:immunity group,post immunity group and control group.After vaccined,we collect tissues at these time:1h,1d,7d,14d,30d(M1),60d(M2),90d(M3),120d(M4),150d(M5),180d(M6),210d(M7),240d(M8),270d(M9),300d(M10),330d(M11),360d(M12).After detected,we found that pCAGGoptiHA5 is detected in all tissues and each time;control group was not found pCAGGoptiHA5; pCAGGoptiHA5 was raised after post immunity; plamid in muscle achieved maximum at 1h, plamid in liver,spleen,kidney,stomach, intestine and brain achieved maximum at Id, plamid in heart and lung achieved maximum at 7d,then decreased gradually;in all tissues,clearance of pDNA from brain was thefastest,1.83 log copies/ug(67copies/ug)at 360d, however,clearance of pDNA from the injection site was the slowest.4.5 log copies/ug(30300copies/ug) at 360d. Inorder to determine whether pDNA integration into the genome of the vaccinated animal.we recovery pCAGGoptiHA5 production and tag DNA probe,after determined probe sensibility we choose tissues of 30d,120d and 360d to detect.The result showed that pDNA did not integration into the genome of the vaccinated animal.It is identical with other studies.This is first time to confirm avian influenza DNA vaccine persist in chicken for a year, defined the distribution and persistence of plamid DNA in chicken and make sure that pDNA did not integrat into the genome of the vaccinated animal.These studies provide theory guidance for H5 avian influenza DNA vaccine and references for Biodistribution and safty studies of DNA vaccine.