Protease susceptibility assay. Each reaction contained 3 ml of the anti-influenza vaccine season 2011-2012 (InflexalV-Crucell), which contains 30 ng of A/California/7/09 (H1N1) HA or 3 ml of the anti-influenza vaccine season 2011-2012 combined with 2 fold molar excess of PN-SIA49. Titron X-100 was added to prevent aggregation of the post-fusion HA. The pH was lowered in all samples except controls using citric acid 0.1 M pH 3. Reactions were mixed, briefly centrifuged and incubated at 37uC for one hour. After incubation, reactions were equilibrated to room temperature and the pH was neutralized by addition of 1 M Tris, pH 9. The actual pH reached was determined in parallel using larger buffer volumes without protein. Trypsin was added to all samples except controls at a final ratio of 1:25 by mass and samples were digested overnight at 37uC. Non-reducing SDS buffer was added to each reaction. Samples were boiled for ,2 minutes and loaded on a nonreducing 4-15% polyacrilamide pre-casted gel (Biorad, Italy). After running, samples were transferred on a PVDF membrane (PerkinElmer, Belgium) for 2 hours at 350 mA. The membrane was then blocked with 5% not fat milk in PBS-Tween20 0,1% (PBST) for 1 h at room temperature and then washed three times with PBST. PN-SIA28, a human anti-HA monoclonal antibody recognizing HA0 [39] , was used as primary antibody at 1 mg/ml in 5% not fat milk-PBST. The membrane was incubated for 1 hour at room temperature and then was washed three times with PBST. Secondary anti-human antibody was added and incubated for 1 h at room temperature. After incubation the membrane was washed, the substrate solution (SuperSignalH West Pico Chemiluminescent Substrate, PIERCE) was added and incubated for 2 min.