| In recent years, attenuated intracellular bacteria have elicited great concerns as vehicles for DNA vaccine delivery. DNA vaccines can be directly delivered into antigen presenting cells (APCs) by this means. It has been reported in many mouse and fish models and has shown the ability to induce strong humoral, cellular and local mucosal immune response for enhancement of DNA uptaking efficacy. But so far, this system has not been tested in avian models. In our experiments, we constructed three types of H5 subtype of Avian influenza virus (AJV) DNA vaccines delivered by attenuated S. typhimurium and S. gallinarum respectively, and evaluated their immunobiological propterties hi mice and chicks.Firstly, a pair of primers were designed and synthesized according to the previously published mRNA sequence in Genebank and used to amplify HA gene by reverse-transcription polymerase chain reaction (RT-PCR) from the genomic RNA of a H5 subtype AFV strain A/Goose/Jiangsu/l/2000(H5Nl) isolated from goose. A desired PCR product of 1.7kb was obtained, and then the PCR product was cloned intopGEM T Easy vector for sequencing. It was shown that its sequence has 97.9% and97.6% of identity to that of HA gene of A /Goose/Guangdong/1/1996(H5N1) and A /Hongkong /156 71997 (H5N1) respectively, and 79.8% and 88.3% of identity to that of North America reference strain A/Chicken/Pennsylvinia/1/83(H5N2) and Eurasian reference strain A/Turkey/Ireland/1378/83(H5N8). These results suggested that this isolate was probably originated from Eurasia. Subsequently, HA gene was subcloned into eukaryotic expression vector pVAX1 and asd-pVAX1 through Hindlll and BamHI double digestion. The recombinant vectors pVAXl-HA and asd-pVAX1-HA were transfected P815 cells, and identified for the transient expression of ATV HA gene in indirect immunofluorescent assay. Finally, the recombinant vectors were transformed into final hosts of attenuated S. typhimurium X4550 and attenuated S. gallinarum 97A, the recombinants were screened and designated as X4550 (pVAX1-HA), X4550(asd-pVAX1-HA) and 97A (pVAX1-HA), respectively. At the same time, this HA gene was subcloned into eukaryotic expressing vector pcDNA3.1(-), and the resulting recombinant vector pcDNA3.1(-)-HA was used to transfect P815 cells. With G418 screening, stably transfected P815 cells expressing HA proteins, named P815-HA , were obtained and identified by indirect immunofluorescent assay.Six weeks old BALB/c mice were immunized with X4550 (pVAXl-HA) and X4550 (asd-pVAXl-HA) at four weeks interval and challenged subcutaneously in abdomen with P815-HA cells four weeks postboosting, all mice from groups of negative control and blank control harboured tumours at injected sites, but all immunized mice were resistant to tumour cell challenge, it suggested that specific CTL effector cells against HA protein were elicited in immunized mice . In two immunized groups, some mice were induced detectable ELISA antibody titers. These results showed that X4550(pVAX1-HA) and X4550 (asd-pVAXl-HA) were immunogenicity for BALB/c mice.In order to determine their dynamic changes in vivo , two weeks old commercial ISA brown chicks were administered orally with three recombinant attenuated Salmonellae carrying DNA vaccines. At different time points, chicks' peripheral blood, spleen, liver, small intestine and caecum samples were collected and inoculated in selective LB plates, the number of CPU were documented. The results showed that vaccinated bacteria mainly resided in caceum. and the bacteria could be isolated from caecum during the 30 days experiment period. All the three bacteria were diffident in the ability to infect internal organs (such as spleen and liver). For X4550(pVAXl-HA) and X4550 (asd-pVAXl-HA). they can only be recovered with low CPU number from spleen and liver during limited time. For 97A(pVAX1-HA), it failed to be isolated at any time point. It suggested that it's a heavy duty for Salmonella hosts to carry and deliver DNA vaccines, but they still have the potency to trigger the immune s...
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