| Vaccination plays an extremely important role in control of avian influenza 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 compromise to maternal antibody and less protective efficacy than inactivated vaccines. It may be one of efficient ways to solve these problems using cytokines as an immune adjuvant. The recombinant fowlpox viurs (rFPV) co-expressing HA gene of H5subtype AIV and chicken interleukin6(chiIL-6) gene was constructed in our lab. In this study, we investigated the biological characteristics, antibody dynamic in different immune routes and immune protection of the recombinant fowlpox virus.1. Biological characteristics of recombinant fowlpox virus (rFPV-AIH5AIL6)The recombinant fowlpox viruses rFPV-AIH5AIL6, rFPV-AIH5A, rFPV-IL6and wild type strain FPV were grown in chicken embryo fibroblast (CEF) and harvested at different time points. The growth curve and sizes of plaque diameters of recombinant fowlpox virus were similar as that of wild type strain. The rFPV-AIH5AIL6was then passaged in CEF for the20generations. The0th,5th,10th,20th generation of rFPV-AIH5AIL6were selected for PCR amplification, sequence, and determination of genes expression. Sequences analysis of HA gene and IL-6gene showed that there was no point mutation in HA gene after CEF passage, while there was one amino acid mutant at the stop code in IL6gene in the20th generation of rFPV-AIH5AIL6, but the mutation did not affect antigen epitope of the protein. The expression of IL6and HA gene in vitro were confirmed by RT-PCR and indirect immunofluorescence assay. The plaques of rFPV-AIH5AIL6for selected four generations were all blue after the agar cover containing X-GAL. These data indicate that rFPV-AIH5AIL6has a good genetic stability.2. Antibody dynamics of SPF chicken immunized with rFPV-AIH5AIL6in two immune routes.Groups of10-day-old SPF chickens were inoculated by subcutaneous route in the back of neck and wings with inoculums containing rFPV-AIH5A, rFPV-AIH5AIL6, rFPV-AIH5AIL2, rFPV-IL6, wt-FPV, PBS, respectively. One group was vaccinated subcutaneously in the back of neck with killed vaccines as a control group. There is no significant difference in body weight between the group of rFPV-AIH5AIL6and the group of normal control, but the body weight in group of wild type strain was lower than that of the group of normal control. The sera were Collected at10,14,21,28,35,42,49days post vaccination. The result of HI antibodies showed that the chickens inoculated subcutaneously by wing route can produced higher level HI antibody. HI antibody induced by rFPV-AIH5AIL6were higher than that induced by rFPV-AIH5AI2ã€rFPV-AIH5A strain. HI antibody reached a peak at21days post vaccination, decreased at28days post vaccination, and remained at a certain level at49days post vaccination.3. Protection trial of chickens immunized with recombinant fowlpox viruses. Groups of10-day-old SPF chickens were inoculated by subcutaneous route in wings with inoculums containing FPV-AIH5A, rFPV-AIH5AIL6, rFPV-AIH5A+rFPV-IL6, rFPV-AIH5AIL2, rFPV-IL6, rFPV-IL2, wt-FPV, PBS. One group of chickens was inoculated with killed vaccine as a positive control. There is no significant difference in body weight between the group of rFPV-AIH5AIL6and the group of normal control. The chicken was challenged with H5subtype avian influenza virus at21days post vaccination. The protection indexes of groups immunized with rFPV-AIH5AIL6, rFPV-AIH5AIL2, and killed vaccine were95%,85%, and95%, respectively, significantly higher than that of other groups. The result of virus shedding showed that the shedding rate of chickens immunized with rFPV-AIH5AIL6was the lowest.In summary. Chicken IL6could improved the immune efficacy against AI when it was co-expressed with HA gene in recombinant fowlpox virus. This study paved the way for further development of a new AIV recombinant vaccine. |
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