Subunit influenza vaccine candidate based on CD154 fused to HAH5 increases the antibody titers and cellular immune response in chickens

World Health Organization has a great concern about the spreading of avian influenza virus H5N1. To counteract its massive spread, poultry vaccination is highly recommended together with biosecurity measures. In our study, a recombinant vaccine candidate based on the fusion of extracellular segments of hemagglutinin (HA) H5 of avian influenza virus and chicken CD154 (HACD) is tested with the aim of enhancing humoral and cellular immune responses in chickens. Protein expression was carried out by transducing several mammalian cell lines with recombinant adenoviral vectors. HACD purification was assessed by three distinct purification protocols: immunoaffinity chromatography by elution at acidic pH or with a chaotropic agent and size exclusion chromatography. Humoral and cellular immune responses were measured using the hemagglutination inhibition assay and the semiquantitative real time PCR, respectively. The results showed that humoral response against HACD was significantly higher than the obtained with HA alone after booster (P<0.01, P<0.05). From HACD molecules purified by distinct protocols, only the obtained by size exclusion chromatography generated hemagglutinationin-inhibition activity. IFN-γ levels indicated that cellular immune response was significantly higher with HACD, in its pure or impure form, compared to its counterpart HA (P<0.01). These data demonstrate that HACD is able to significantly enhance humoral and cellular immune responses against HA antigen, which make this fusion protein a promising subunit vaccine candidate against H5N1 virus outbreaks.