| Vaccination plays critical role in control of avian influenza. However, conventional inactivated vaccines have some disadvantages with high cost and interference with surveillance. Therefore, development of a new vaccine is in urgent need. Genetic engineering vaccines based on fowlpox virus vector expressing protective antigenic genes of avian influenza virus have been well established and developed, but all current recombinant fowlpox vaccines show the sensitivity to maternal antibody interference and less protective efficacy than that of inactivated vaccines. It may be one of efficient ways to solve these problems using cytokines as the immune adjuvant. In order to develop genetic engineering vaccines which are safe, convenient, and more effective for control of avian influenza, we designed a series of experiments to reach the goal.1. Construction of recombinant fowlpox viurs (rFPV) co-expressing the HA gene of H5 subtype AIV and the chicken interleukin 2 (chiIL-2) gene The chicken interleukin 2 (chiIL-2) gene were inserted into expressing vector p12LS to construct a recombinant transferring vector p12LSIL2. The hemagglutinin (HA) gene from H5N1 avian influenza virus and the chicken interleukin 2 (chiIL-2) gene were inserted into expressing vector p12LS to construct recombinant transferring vector p12LSH5AIL2, in which HA gene regulated by promoter Ps was inverse tandem connection with chiIL-2 gene regulated by promoter PE/L. Then the p12LSIL2 and p12LSH5AIL2 were used to transfect chicken embryo fibroblasts (CEF), which was pre-infected with wild type fowlpox virus. The rFPV-IL2 expressing chiIL2 gene and rFPV-AIH5AIL2 coexpressing HA and chiIL2 genes were obtained and purified by selection of blue plaques on the CEF. The expression of HA gene in vitro by rFPV-AIH5AIL2 was confirmed by indirect immunofluorescence assay, and the expression of chiIL2 gene by rFPV-IL2 and rFPV-AIH5AIL2 were confirmed by RT-PCR and indirect immunofluorescence assay.2. Construction of recombinant fowlpox viurs (rFPV) co-expressing the HA gene of H5 subtype AIV and the chicken interleukin 6 (chiIL-6) geneThe chicken interleukin 6 (chiIL-6) gene was synthesized artificially, pieced together, and then inserted into p12LS to form recombinant transferring vector p12LSIL6 and p12LSH5AIL6. The p12LSIL6 and p12LSH5AIL6 were used to transfect chicken embryo fibroblasts (CEF), which was pre-infected with wild type fowlpox virus.The rFPV-IL6 expressing chiIL6 gene and rFPV-AIH5AIL6 coexpressing HA and chiIL6 genes were obtained and purified by selection of blue plaques on the CEF. The expression of HA gene in vitro by rFPV-AIH5AIL6 was confirmed by indirect immunofluorescence assay, and the expression of chiIL6 gene by rFPV-IL6 and rFPV-AIH5AIL6 were confirmed by RT-PCR.3. Vaccination trials in chickensTen groups of 7-day-old SPF chickens were inoculated by subcutaneous route in the base of neck with inoculums containing rFPV-HA, rFPV-HA and rFPV-IL2, rFPV-HA and rFPV- IFN-â…¡, rFPV-AIH5AIL2, respectively. Other groups were vaccinated with kill vaccination, wt-FPVLP, rFPV-IL2 and PBS as the control. The commercial chickens with high titer of maternal antibody against AI were immunized with the same vaccines as in SPF chickens. Experiments on SPF and commercial chickens demonstrated that HI antibody induced by rFPV-AIH5AIL2 strain were significantly higher than that induced by rFPV-HA strain. The Chickens immunized with rFPV-AIH5AIL2 strain produced similar ratios of protective efficacy and virus shedding as the one immunized with rFPV-HA strain in SPF chicken. However, in commercial chicken, the chickens immunized with rFPV-AIH5AIL2 strain produced significant higher protection and lower virus shedding against H5N1 avian influenza virus challenge than the one immunized with rFPV-HA strain. These results indicated chiIL2 could improved the immune efficacy against AI when it was coexpressed with protective antigen in recombinant fowlpox virus. This study paved the way for further development of a new AIV recombinant vaccine. |
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