| Domestic ducks play an important role in the epidemiology of avian influenza. In order to prevent and control avian influenza in ducks more effectively, researchers are working to develop improved genetic engineering vaccines to enhance the immune efficacy of vaccines. Genetic engineering vaccines based on fowlpox virus vector have been well established and developed. As it is known that fowlpox virus can be used as both replicative and nonreplicative vectors. Although fowlpox virus vectored vaccine has a number of advantages, such as added safety and low interference by maternal antibody, while being used as nonreplicative vector, it requires a high dose administration with boosting injections to achieve sufficient protection. Cytokines have been proven to be effective immunomodulator and it may be one of efficient ways to improve the immunogenicity of nonreplicative vector-based vaccines. Previous study showed that the two recombinant fowlpox viruses co-expressing the HA gene of H5N1avian influenza virus and the chicken interleukin2(ChIL-2) gene and co-expressing the HA gene of H5N1avian influenza virus and the chicken interleukin6(ChIL-6) gene could provide good immune protection in chickens. In our study we evaluated the immune efficacy of the two recombinant fowlpox viruses in ducks and paved the way for further development of AIV vaccine in waterfowl.1. Immune efficacy of recombinant fowlpox viruses co-expressing the HA gene of H5N1avian influenza virus and ChIL-2gene or ChIL-6gene in mallard ducksA total of14424-day-old mallard ducks were divided randomly into8groups. The ducks were immunized with105PFU (0.2mL per duck) of rFPV-SYHA, rFPV-LKHA, rFPV-AIH5AIL6, rFPV-AIH5AIL2, rFPV-IL6, and wt-FPV,0.5mL of H5AI killed vaccine and0.2mL of sterile PBS, respectively. The body weight gain of all ducks were measured and the sera of each group were collected at days0,7,14and21post vaccination. Antibody titers of sera were determined by HI assay. The thymuses, spleens, bursas, and peripheral blood of each group were collected at days0(PBS inoculated group only),9,16and23post vaccination for lymphocyte proliferation assay and erythrocyte rosette test. At days23post vaccination, each duck was challenged with0.2mL of105.75EID50H5subtype HPAI virus (A/mallard/Huadong/S/2005) by nose/eye dropping. Furthermore, tracheal and cloacal swabs were collected for virus isolation from each group at days3,5and7post challenge.We found that the antibody titers in rFPV-AIH5AIL6and rFPV-AIH5AIL2immunized ducks were higher than that in rFPV-SYHA injected ducks, and significantly lower than that in ducks immunized with H5killed vaccine. The lymphocyte proliferation levels as well as the active and total E-rosette rates of rFPV-AIH5AIL6and rFPV-AIH5AIL2immunized ducks were higher than that of other rFPVs vaccinated ducks. The survival rates of mallard ducks inoculated with rFPV-AIH5AIL6and rFPV-AIH5AIL2were80%and66.7%, respectively, higher than that of ducks immunized with rFPV-SYHA and rFPV-LKHA (41.7%and33.3%, respectively). We also observed the lower shedding rates and shorter shedding duration in rFPV-AIH5AIL6and rFPV-AIH5AIL2immunized mallard ducks. Furthermore, the rFPVs have no side effect on body weight gain in mallard ducks.2. Immune efficacy of recombinant fowlpox viruses co-expressing the HA gene of H5N1avian influenza virus and ChIL-2gene or ChIL-6gene in cherry valley meat duckA total of14410-day-old cherry valley meat ducks were divided randomly into8groups. The ducks were immunized with105PFU (0.2mL per duck) of rFPV-SYHA, rFPV-AIH5AIL6, rFPV-AIH5AIL2, rFPV-IL6, rFPV-IL2, rFPV-IL6, and wt-FPV,0.5mL of H5AI killed vaccine and0.2mL of sterile PBS, respectively. The sera of each group were collected at days0,7,14and21post vaccination. Antibody titers of sera were determined by HI assay. The thymuses, spleens, bursas, and peripheral blood of each group were collected at days0(PBS inocualted group only),7,14and21post vaccination for lymphocyte proliferation assay and erythrocyte rosette test. The spleens of each group were collected at days19post vaccination used for detecting the expression level of MHC Ⅰ, MHC Ⅱ and RIG-Ⅰ genes by quantitative real-time RT-PCR. The body weight of each duck were measured at days0and21post immunization. At days21post vaccination, each duck was challenged with0.2mL of105.75EID50H5subtype HPAI virus (A/mallard/Huadong/S/2005) by nose/eye dropping. Tracheal and cloacal swabs were collected for virus isolation from each group at days3,5and7post challenge.We found that the antibody titers in ducks induced by rFPV-AIH5AIL6and rFPV-AIH5AIL2were higher than that induced by rFPV-SYHA, and significantly lower than that induced by H5killed vaccine. The lymphocyte proliferation levels as well as the active and total E-rosette rates of rFPV-AIH5AIL6and rFPV-AIH5AIL2immunized meat ducks were higher than that of other rFPVs vaccinated ducks. The expression level of MHC I in rFPV-AIH5AIL2immunized ducks was significantly higher than that in other ducks. The expression level of MHC Ⅱ in rFPV-AIH5AIL6and H5killed vaccine vaccinated ducks was significantly higher than that in rFPV-SYHA immunized ducks. The rFPV-AIH5AIL6, rFPV-AIH5AIL2and H5killed vaccine immunized ducks all had a high expression level of RIG-I. The post-challenge survival rates of cherry valley meat ducks inoculated with rFPV-AIH5AIL6and rFPV-AIH5AIL2were90%and81.8%, respectively, higher than that immunized with rFPV-SYHA (54.5%). We also observed the lower shedding rate and shorter shedding duration in rFPV-AIH5AIL6and rFPV-AIH5AIL2immunized cherry valley meat ducks. Furthermore, the rFPVs have no side effect on body weight gain in cherry valley meat ducks.In conclusion, these results suggested that ChIL-2and ChIL6may be effective adjuvants for recombinant fowlpox virus vectored vaccine to promote cellular and humoral immune response, improve the protection efficacy and inhibit virus shedding in ducks. |
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