New antigen against avian influenza virus based on the fusion of the exposed domains of the H5 subtype hemagglutinin and the Gallus gallus CD154 protein

The highly pathogenic H5N1 avian infl uenza virus (AIV) has caused important economic losses and public health problems, mainly in Asia, Africa and Europe. Counteracting this virus by vaccination of poultry is crucial. In our study, recombinant antigens based on the fusion of the extracellular segments of both molecules, hemagglutinin H5 of AIV and the chicken CD154, were tested to enhance the immune response in chickens. For this purpose, chickens were immunized with recombinant adenoviral vectors carrying the nucleotide sequence of the hemagglutinin H5 alone (AdHA) or fused to the CD154 recombinant protein (AdHACD). Also, these two proteins (rHA and rHACD) were produced and assayed as antigens. Both, the adenoviral vector AdHACD and the recombinant protein rHACD, induced hemagglutination-inhibiting antibody titers signifi cantly higher than their counterparts carrying HA alone. Similar results were observed when the cellular response was measured by real time PCR. AdHACD and rHACD signifi cantly increased the production of IFN-γ transcripts, which were lower when AdHA or rHA were used. To obtain a higher quality antigen, rHACD was purifi ed by size exclusion chromatography, resulting in the preservation of its immunogenic properties. However, rHACD did not work as immunogen when an immunoaffi nity chromatography was performed by eluting at acidic pH or with a chaotropic agent. These results demonstrate that chicken CD154 can signifi cantly enhance humoral and cellular responses against hemagglutinin H5 of AIV when both molecules are administered as a fusion protein in chickens. Therefore, AdHACD or rHACD could become promising vaccine candidates against H5N1 virus.