Abstract
Avian influenza (AI) has posed a serious threat to the poultry industry and public health, and has resulted in tremendous economic and social impacts in the last few decades. Avian influenza viruses (AIVs) are widespread in domestic and wild birds due to their highly contagious property and frequent adaptive evolution. To prevent and control the AIVs, vaccination is one of the most effective ways. As for the antigens being used in vaccines, virus-like particles (VLPs) are promising candidates due to their native and non-infective natures. Here, we developed a trivalent VLP displaying the hemagglutinin (HA) of H5, H6 and H7 AIVs, the most common AIV subtypes found in Taiwan and other countries. Full-length HA protein genes of AIVs H5N6, H6N1, H7N9 and the matrix protein 1 (M1) gene of AIV H6N1 were cloned onto baculoviral vectors separately, and the trivalent VLPs (H567-M1 VLPs) were then generated by co-infecting cells with three individual recombinant baculoviruses. On the other hand, full length of HA protein genes and gag protein gene from the bovine leukemia virus were cloned onto the baculoviral vector, and the trivalent VLPs (H567-Bgag VLPs) were then generated by infecting insect cells with one single recombinant baculoviruses. The results showed that, use of Spodoptera frugiperda 21 (Sf21) culture for infection improved the expression yield of trivalent VLPs, as compared with the HighFive cells. The expression of H567-Bgag VLPs was more stable than that of H567-M1 VLPs. Furthermore, immunofluorescence assay was used to determine H5/HA, H6/HA and H7/HA expression in Sf21 cells and each subtype were expressed successfully. Then, VLPs were purified from the Sf21 culture supernatant by anion exchange chromatography. The morphology of VLPs was confirm by TEM. The size of H567-M1 VLPs were 91 nm and that of H567-Bgag VLPs’ was 145 nm. The hemagglutination activity of H567-M1 VLPs and H567-Bgag VLPs were 64 HAU per 25 l and 8192 HAU per 25 l after purification. In summary, we developed a versatile VLP platform displaying HA antigens of multiple subtypes. Multivalent VLP antigens with high quality are anticipated to show promises in the application of vaccine development for disease control.
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