Figure 1

B-COBRA HA antigens for the vaccination and infection of pre-immune ferrets. (A) Publicly available HA sequences from 1940 to 2011 were downloaded and used to design three unique influenza B HA antigens: BC1, a single consensus sequence of 318 sequences during this period; BC2, the same 318 sequences antigenically layered into 10 secondary sequences and then a final consensus sequence; BC3, 217 sequences from 1999 to 2011 layered into 5 secondary sequences and then a final consensus sequence. (B) Phylogenetic analysis of B-COBRA candidates along with current and past IBV vaccine strains. HA1 regions were extracted and aligned using Muscle 3.8.425. The tree was rendered using FastTree 2.1.11. B/YAM lineage shown with yellow font, B/Victoria-lineage with violet font, and B-COBRA candidates in red font. (C) Three plasmids containing genes for HA, NA, or a Gag viral matrix protein were lipotransfected into mammalian 293T cells. After 3–5 days, supernatants were collected, centrifuged, filter-sterilized, and then ultracentrifuged with a 20% glycerol cushion. Pellets were resuspended in a small volume of PBS, and then HA activity and protein concentration were determined. (D) Immunologically naïve ferrets were infected intranasally (day 0) with a B/YAM (SH/02) or B/VIC (HK/01) virus and then vaccinated intramuscularly once (day 90) with one of three B-COBRA VLP vaccines (BC1, BC2, BC3), one of four VLP vaccines expressing wild-type HA proteins from the B/VIC lineage (MY/04 or CO/17) or B/YAM lineage (FL/06 or PH/13), or a mock vaccination containing PBS and adjuvant. All vaccines were adjuvanted 1:1 with a squalene-based emulsion adjuvant. Blood was collected from all animals at days 0, 90, and 120. All ferrets were challenged with an IBV heterologous to their pre-immune infection and nasal washes were collected 3 days post-infection. Created with BioRender.com.