The HA ectodomain-encoding cDNA was cloned into the pCD5 expression vector for efficient expression in mammalian cells [9]. The pCD5-Cal/04/09 vector had been modified such

that the HA-encoding cDNA was cloned in frame with DNA sequences coding for a signal sequence, a GCN4 isoleucine zipper trimerization motif (KRMKQIEDKIEEIESKQKKIENEIARIKK) [10] and the Strep-tagII (WSHPQFEK; IBA, Germany). The HA ectodomain was expressed in HEK293T as previously described [11]. HA protein expression and secretion was confirmed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by western blotting using a mouse anti-Strep-tag antibody (IBA, Germany). Secreted HA proteins were purified SB431542 cost using Strep-tactin sepharose beads according to the manufacturer’s instructions (IBA, Germany). The concentration of purified protein was determined by

using a Nanodrop 1000 spectrophotometer (Isogen Life Sciences) according the manufacturer’s instructions. Oligomeric status of the HA protein was determined by analyzing the elution profile using a Superdex200GL 10–300 column and by blue-native gel-electrophoresis. The vaccine was formulated with Specol [12] and [13] as an adjuvant, at 25 μg HA per dose of 2 ml. Pigs were vaccinated intramuscularly. Influenza virus A/Netherlands/602/2009 (H1N1)v was isolated from the first confirmed case in the Netherlands [14]. The patient was a 3-year old boy, developing a fever and symptoms of HKI 272 respiratory disease after returning from Mexico with his family. A nasal swab was taken before the patient was treated with oseltamivir. Virus was initially grown on embryonated eggs, and subsequently passaged on Madin–Darby canine kidney (MDCK) cells before it was used to inoculate the pigs. This virus differs by 8 amino acids from the A/California/4/2009 Florfenicol (H1N1)v strain [14]. Because it is, however, closer to the consensus sequence, it is considered representative of the circulating H1N1v influenza strains. Pigs were inoculated with a dose of 107.5 TCID50, suspended in 2 ml PBS, of which 1 ml was nebulised within

each nostril. Clinical symptoms and body temperature were recorded daily from day 3 before inoculation until the end of the experiment. At days 1–3 p.i. clinical symptoms and body-temperature were recorded twice per day with a 12 h interval. Serum samples were collected during both times of vaccination, at the time of inoculation, and 7, 10, 14 and 21 days p.i. Oropharyngeal and nasal swabs were collected daily from all animals still alive from day 0 to 11 p.i., and on days 14, 17 and 21 p.i. For oropharyngeal swabs multi-layered gauze dressings in a pair of tweezers were used to scrape the palatine tonsils at the dorsal pharyngeal wall, behind the soft palate. Nasal swabs were collected using sterile rayon swabs (Medical Wire & Equipment, Corsham, United Kingdom).