Intranasal vaccination with H5, H7 and H9 hemagglutinins co-localized in a virus-like particle protects ferrets from multiple avian influenza viruses

• Recombinant triple-subtype VLPs were made, which co-localized H7, H5, and H9 proteins in the VLP.
• Triple-subtype VLPs were expressed, characterized, and isolated from Sf9 cells.
• Recombinant triple-subtype VLP vaccine was administered to ferrets intranasally.
• Vaccinated ferrets were challenged with live pathogenic H7, H5, and H9 viruses.
• Protection of ferrets from infection with three avian influenza viruses was demonstrated.

Avian influenza H5, H7 and H9 viruses top the World Health Organization's (WHO) list of subtypes with the greatest pandemic potential. Here we describe a recombinant virus-like particle (VLP) that co-localizes hemagglutinin (HA) proteins derived from H5N1, H7N2, and H9N2 viruses as an experimental vaccine against these viruses. A baculovirus vector was configured to co-express the H5, H7, and H9 genes from A/Viet Nam/1203/2004 (H5N1), A/New York/107/2003 (H7N2) and A/Hong Kong/33982/2009 (H9N2) viruses, respectively, as well as neuraminidase (NA) and matrix (M1) genes from A/Puerto Rico/8/1934 (H1N1) virus. Co-expression of these genes in Sf9 cells resulted in production of triple-subtype VLPs containing HA molecules derived from the three influenza viruses. The triple-subtype VLPs exhibited hemagglutination and neuraminidase activities and morphologically resembled influenza virions. Intranasal vaccination of ferrets with the VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with H5N1, H7N2, and H9N2 viruses.