ABSTRACT
Avian influenza virus H5N1 has caused extensive damage worldwide among poultry and humans. Effective expression systems are needed for the production of viral proteins required for monitoring this devastating disease. The present study deals with the establishment of a stable expression system for the hemagglutinin H5 (HAH5) of avian influenza virus using CHO cells in suspension culture transduced with a recombinant lentiviral vector. The synthetic gene coding the HAH5 protein was inserted in a lentiviral vector with the aim of performing a stable transduction of CHO cells. After the selection of recombinant clones, the one with the highest expression level was adapted to suspension culture and the HAH5 protein was purified by immunoaffinity chromatography from the culture supernatant. There were no significant differences when this protein, purified or direct from the culture supernatant of CHO or SiHa cells, was utilized in an immunologic assay using positive and negative sera as reference. It was also demonstrated that the HAH5 protein in its purified form is able to bind anti-HAH5 antibodies generated with proper and non-proper folded proteins. The results demonstrate that the CHO cell line stably transduced with a lentiviral vector coding the sequence of the HAH5 protein and cultured in suspension can be a suitable expression system to obtain this protein for diagnostic purpose in a consistent and reliable manner.
ABSTRACT
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.
