Achievement of avian influenza virus-like particles that could be used as a subunit vaccine against low-pathogenic avian influenza strains in ducks.

Infections with H5/H7 low-pathogenic avian influenza (LPAI) viruses are now notifiable because such viruses can mutate into highly pathogenic avian influenza viruses, leading to serious problems for both animal and public health. Domestic ducks can play a crucial role in the transmission of H5 LPAI viruses to other poultry. Although prime boost vaccination using, respectively, a recombinant vaccine and an inactivated vaccine was shown to be protective in ducks against H5N1 highly pathogenic avian influenza, vaccination of domestic ducks against H5 LPAIV is poorly documented. However, substituting inactivated vaccines with subunit vaccines might be more advantageous. In this context, we generated a triple recombinant baculovirus composed of HA and NA proteins derived from a French H5N3 LPAI virus strain and the M protein derived from an Italian H7N1 LPAI virus strain. We describe a molecular construction strategy that enabled the development of virus-like particles (VLPs). Western blot analyses and neuraminidase inhibition assay of cell supernatants purified by sucrose density gradient ultracentrifugation showed that HA, NA and M1 proteins were expressed and co-released. Electron microscopy examination revealed VLPs that were morphologically identical to wild-type virus. Immunogold electron microscopy demonstrated that H5 and N3 proteins were present on the VLP surface, and haemagglutination and neuraminidase assays showed that the H and N proteins, respectively, were biologically active. In addition, VLP immunogenicity (induction of haemagglutination-inhibiting antibodies) was demonstrated in specific pathogen free Muscovy ducks. According to our successful previous experimental results of protection in ducks following vaccination with the three baculovirus-expressed proteins, the present results make feasible the reliable use of H5N3 VLPs as a subunit vaccine in this species.