*2.2. DNA Expression Adjuvant Constructs and Immunizations*

pFliC-Tm(-gly) *S. typhimurium* has been described previously [25]. pFliC-Tm(-gly) was subjected to site-directed mutagenesis to insert two in-frame translational stop-codons after AA 459 of FliC(-gly) to generate a secreted version of FliC(-gly) (AA numbering is based on GenBank Accession #D13689). Changes were confirmed by DNA sequencing. The immunizations were intranasaly performed (i.n.) as previously described (Table 1) with five groups of outbred female NMRI mice and five groups of inbred female C57BL/6J mice. Briefly, mice were sedated with isoflurane (4% in air) and given WIV vaccine 5 μL/nostril (total volume 10 μL/mouse, 1.5 μg HA antigen/mouse or a total of 25 μg protein/mouse). In groups where the N3 adjuvant was used, a 1% concentration was intranasally given, 6 uL/nostril. Female mice of the NMRI strain were purchased from ScanBur, Sollentuna, Sweden, and C57BL/6 mice were purchased from Charles River, Dortmund, Germany. The animals were kept until 13 (±1) months before immunizations were initiated in accordance with ethical guidelines and permissions at the AF animal facility at the Karolinska Institutet, Stockholm, Sweden under specific pathogen free conditions [26].


**Table 1.** Study design and nasal immunization of NMRI and C57BL/6 mice against formalin-inactivated Influenza A/H1N1/Salomon Island/2006.

Abbreviations: HA = Hemagglutinine protein from influenza A, pFliC(-gly) = Plasmid encoding secreted Flagellin type C of *Salmonella typhimurium* with mammalian glycosylation signal sequences removed.
