Fig. 1
Timeline for vaccine administration. Female BALB/c mice (18–22 months of age) were vaccinated twice on day 0 (d0) and day 21 (d21) with plant-derived H1-VLP vaccine, inactivated H1N1 split vaccine or PBS. Three groups of animals received the VLP vaccine: i) two 3 μg doses intramuscularly (IM/IM) ii) a first 3 μg dose IM boosted at d21 by a 3 μg dose intranasally (IM/IN: Prime-Pull group) or iii) two doses of 1.5 μg IM plus 1.5 μg IN (IM + IN: Multi-modality group). The comparator group received two 3 μg doses of a split inactivated influenza vaccine. Peripheral blood was collected at d0 (pre-vaccination) and at d21 (data not shown) and d42 after the first dose of vaccine. Spleens and lungs were harvested from individual animals. The remaining mice (typically 6–8 mice/group) were scored for frailty on d40–42 then challenged with a sub-lethal dose of wild-type H1N1 A/California/07/2009 virus (525 TCID50 in 50 μL) by IN instillation (25 μL/nare). Weight loss was monitored daily for up to 28 days. At d45 or 3 days post-infection (dpi), 3–5 mice/group were sacrificed (isoflurane/CO2) and serum (cardiac puncture) and lungs were collected. At d67 (25 ± 4 dpi), surviving mice were scored for frailty and sacrificed to collect serum and lungs