All genes used for recombinant proteins and pseudoviruses were synthesized using mammalian preferred codons. The HA-ferritin fusion gene was generated by fusing the ectodomain of HA (residues HA1 1-HA2 174, H3 numbering system) to H. pylori ferritin (residues 5–167) with a Ser-Gly-Gly linker. Recombinant proteins were produced by transient transfection of expression vectors in 293F cells (Invitrogen) and purified by chromatography techniques (see Methods for detail). The TIV used in this study were 2006–2007 and 2011–2012 Fluzone® (Sanofi Pasteur). Animal experiments were carried out in accordance with all federal regulations and NIH guidelines. Mice were immunized intramuscularly twice with 0.17 μg (Fig. 2a and b) or 1.67 μg (Fig. 2d) of HA-nanoparticles (HA amount) or matched amount of TIV with or without Ribi adjuvant system (Sigma) or with MF59 (Novartis) at a 3-week interval. Ferrets were immunized intramuscularly with 2.5 μg of HA-nanoparticles or 7.5 μg of TIV with Ribi at weeks 0 and 4. H1N1 virus challenge was performed five weeks after the last immunization with 106.5 50% egg infectious dose (EID50) of 2007 Bris virus via intranasal inoculation. Statistical analyses were performed using Prism 5 (GraphPad Software).
Full Methods and any associated references are available in the online version of the paper.