This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals, National Research Council (Eighth Edition, 2011). All mouse procedures were approved by Institutional Animal Care and Use Committee (IACUC) of the Centers for Disease Control and Prevention and performed in accordance with the IACUC guidelines (Protocol # 1619: “Studies on the Pathogenesis of and Immunity to Influenza Viruses in Mice”).
We previously described the molecular cloning of a human monoclonal antibody Fab fragment named PN-SIA49 
. The whole antibody molecule was obtained using the BD BaculoGold System (BD Biosciences Pharmingen, San Diego, CA, USA). Briefly, nucleotide sequences codifying heavy and light chains of the PN-SIA49 Fab fragment were cloned into the baculovirus expression vector pAc-κ-Fc (PROGEN Biotechnik GmbH, Heidelberg, Germany). Sf9 insect cells (Invitrogen, Carlsbad, CA, USA) were co-transfected with the linearized baculovirus DNA (BD Biosciences Pharmingen, San Diego, CA, USA) and the pAc-κ-Fc/PN-SIA49. The obtained virus was inoculated at a multiplicity of infection (MOI) of 5 to infect 1×109 High Five insect cells (Invitrogen, Carlsbad, CA, USA) in a final volume of 1 liter. After an incubation of 96 hours the culture media was collected, clarified by centrifugation and filtered with 0.2 µm filter (Millipore, Billerica, MA, USA). The media was loaded into a protein G column (Amersham Biosciences GE Healthcare, Zurich, Switzerland), the antibody was eluted with citric acid 0.1 M, pH 3, and immediately neutralized with Tris Base 1 M, pH 9. The solution containing the antibody was dialyzed against PBS and then concentrated using Amicon Ultra-15 Centrifugal Filter Devices (Millipore, Billerica, MA, USA). Antibody concentration was determined by SDS-PAGE gel and by spectrophotometric measurement at 280 nm.
An anti-influenza A antibody directed against H1N1 subtype HA, named RB62, and an anti-HCV E2 glycoprotein antibody, named e137 
, produced and purified with an identical procedure were used as controls in all experiments.
Viruses and cells
The following human H1N1 and H3N2 reference strains were acquired from the American Type and Culture Collection (ATCC): A/Puerto Rico/8/1934 (H1N1) (ATCC n. VR-1469); A/Wilson Smith/1933 (H1N1) (ATCC n. VR-1520); A/Malaya/302/1954 (H1N1) (ATCC n. VR-98); A/Hong Kong/8/1968 (H3N2) (ATCC n. VR-544); A/Aichi/2/1968 (H3N2) (ATCC n. VR-547); A/Port Chalmers/1/1973 (H3N2) (ATCC n. VR-810). The swine origin influenza virus (S-OIV) A/Milan/UHSR1/2009 (H1N1v) was previously isolated in our laboratory 
. A swine strain, A/swine/Parma/1/1997 (H1N1), was kindly provided by the Zooprophylactic Institute of Brescia, Italy. All these viruses were tested in the BLS3 laboratory of the Vita-Salute San Raffaele University. The following H1N1, H2N2, H3N2, H5N1, H7N2 and H9N2 viruses were tested at the Centers for Disease Control and Prevention (CDC) of Atlanta, Georgia, USA: A/Wilson Smith/1933 (H1N1); A/Ann-Arbor/6/1960 (H2N2); A/Hong Kong/8/1968 (H3N2); A/Pilippines/01/1982 (H3N2); A/England/648/1989 (H3N2); A/Fukui/20/2004 (H3N2); A/Washington/01/2007 (H3N2); A/NewHampshire/01/2009 (H3N2); A/Vietnam/1203/2004 (H5N1) clade 1; A/duck/Vietnam/NCVD98/2007 (H5N1) clade 2.3.4; A/New York/107/2003 (H7N2); LAIV A/chicken/Hong Kong/G9/1997 (H9N2).
All viruses, excluding the A/swine/Parma/1/1997, were cultured on Madin-Darby Canine Kidney (MDCK) (ATCC CCL-34) cells propagated in Modified Eagle Medium (MEM) (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 2% bovine serum albumin (Gibco Invitrogen, Carlsbad, CA, USA), 50 µg/ml of penicillin (Gibco Invitrogen, Carlsbad, CA, USA), 100 µg/ml of streptomycin (Gibco Invitrogen, Carlsbad, CA, USA) and 2 µg/ml TPCK–trypsin (Roche Applied Science). The A/swine/Parma/1/97 isolate was analogously grown on Newborn Swine Kidney (NSK) cells, kindly provided by the Zooprophylactic Institute of Brescia, Italy. At 80% confluence, cells in MEM supplemented with 2 µg/ml serum-free TPCK-trypsin, were infected with each strain at a MOI of 0.001. After 1 hour of infection, cells were washed with phosphate buffered saline (PBS); MEM supplemented with 2 µg/ml TPCK-trypsin was then added and cells were incubated at 37°C in 5% CO2 atmosphere. Cells were observed daily to monitor the cytopathic effect and, usually after 72–96 hours, the supernatant was collected, centrifuged at 1000 rcf for 10 minutes to eliminate cells debris and filtered with 0.22 µm filters (Millipore, Billerica, MA, USA). The supernatant was then aliquoted and stored at −80°C as cell-free virus.
Virus neutralization assays
Fluorescence inhibition assay and plaque reduction assay
The following H1N1 and H3N2 viruses were tested using the fluorescence inhibition assay: A/Puerto Rico/8/1934 (H1N1); A/Wilson Smith/1933 (H1N1); A/Malaya/302/1954 (H1N1); A/Milan/UHSR1/2009 (H1N1v); A/swine/Parma/1/1997 (H1N1); A/Hong Kong/8/1968 (H3N2); A/Aichi/2/1968 (H3N2); A/Port Chalmers/1/1973 (H3N2). Each viral isolate was titrated by the limiting dilution method and the viral titer calculated by the Reed-Muench formula. Neutralizing assays were carried out in 96 wells plate using MDCK cells (4×104cells/well). Serial dilutions, 10 µg/ml-0.03 µg/ml, of IgG PN-SIA49 were preincubated for 1 hour at 37°C with 100 median tissue culture infective doses (TCID50) of virus. Following incubation, 100 µl of the mix antibody-virus were added to the cells and incubated for another hour at 37°C in 5% CO2. At the end of this incubation, cells were washed with PBS and 100 µl of MEM TPCK-Trypsin (2 µg/ml) were added in each well. Cells were incubated for 7 hours at 37°C in 5% CO2 and then washed with PBS, fixed and permeabilized with ice-cold ethanol. Cells were incubated with anti-influenza A mouse antibody (Argene, Shirley, NY, USA) for 30 minutes at 37°C in a humid chamber. The cells were then washed with PBS and incubated for 30 minutes at 37°C in a dark humid chamber with a FITC-conjugated secondary antibody (Argene, Shirley, NY, USA). Nuclei staining was obtained with Hoechst 33342 (Sigma Aldrich). An infection control without antibody was included, as well as a negative control with the anti-HCV/E2 antibody e137. Each neutralization assay was performed in triplicate and repeated in two different sessions.
The neutralization activity for each antibody concentration was expressed as the percentage reduction of fluorescent nuclei compared with the nuclei count in the infection control. Nuclei counting was performed by using the GE Healthcare's IN Cell Analyzer 1000, an automated epifluorescence based microscope system. The neutralization curves were then fit by non-linear regression with the GraphPad Prism software, allowing IC50 calculation.
The A/PR/8/34 (H1N1) and A/Milan/UHSR1/2009 (H1N1) viruses were also tested in plaque reduction assay as previously described 
. Briefly, neutralizing assays were carried out in 6 wells plates using MDCK cells (5×105 cells/well). Two dilutions, 1-0.1 µg/ml, of IgG PN-SIA49 were preincubated for 1 hour at 37°C with 100 TCID50 of virus. Following this incubation, 1 ml of each virus-antibody mix was added on MDCK monolayer and the plate was incubated 1 hour at 37°C in 5% CO2. Then, the medium was removed and the monolayer washed twice with PBS. Two ml of MEM-agarose 0.8% supplemented with penicillin (50 µg/ml) (Gibco Invitrogen, Carlsbad, CA, USA), streptomycin (100 µg/ml) (Gibco Invitrogen, Carlsbad, CA, USA), L-glutamine (2 mM) (Gibco Invitrogen, Carlsbad, CA, USA) and trypsin (2 µg/ml) (Roche Applied Sciences) were added to each well and the plates were incubated 48 hours at 37°C in 5% CO2. After this incubation, the agarose medium was removed from each well and 1 ml of 70% methanol-crystal violet 1% (w/v) was added to each well at room temperature. Finally, the wells were washed with tap water and dried. An infection control without antibody was added as well as a negative control with anti-HCV/E2 e137 mAb. The neutralization was determined counting the PFU reduction in presence of antibodies compared to the infection control.
Infectious foci formation reduction assay
The following H1N1, H2N2, H3N2, H5N1, H7N2 and H9N2 viruses were tested: A/Wilson Smith/1933 (H1N1); A/Ann-Arbor/6/1960 (H2N2); A/Hong Kong/8/1968 (H3N2); A/Pilippines/01/1982 (H3N2); A/England/648/1989 (H3N2); A/Fukui/20/2004 (H3N2); A/Washington/01/2007 (H3N2); A/NewHampshire/01/2009 (H3N2); A/Vietnam/1203/2004 (H5N1) clade 1; A/duck/Vietnam/NCVD98/2007 (H5N1) clade 2.3.4; A/New York/107/2003 (H7N2); LAIV A/chicken/Hong Kong/G9/1997 (H9N2). Each viral isolate was titrated to establish working dilution that produces 15-30 foci forming units per well in 96 tissue culture plates. Neutralizing assays were carried out in 96 wells plate using MDCK/SIAT-1 cells. Serial dilutions, 30 µg/ml-0.37 µg/ml, of IgG PN-SIA49 were preincubated for 1 hour at 37°C with the subset of viruses. Following this incubation, 100 µl of the antibody-virus mix was added to the cells and incubated for another hour at 37°C in 5% CO2. At the end of this incubation, the cells were washed twice in PBS and 100 µl of virus growth media containing 2 µg/ml of TPCK treated trypsin was added. Cells were incubated for 12–16 hours at 37°C in 5% CO2 and then washed with PBS, fixed and permeabilized with ice cold methanol/acetic acid (95
5) for 30 min at −20°C. Cells were incubated with anti-NP antibodies (Millipore, Billerica, MA, USA) for 30 minutes at 37°. The cells were then washed and incubated for 30 minutes at 37°C with a mouse HRP-conjugated secondary antibody. True Blue chromogenic substrate (KPL) was used to count the number of foci.
Murine lethal challenge models
Female BALB/c mice were purchased at 6 to 8 weeks of age from Charles River Co. (Wilmington, MA). All mice were maintained in specific pathogen–free barrier facilities. All animal experiments and procedures conformed to protocols approved by the Centers for Diseases Control and Prevention (CDC), Atlanta, GA, USA.
For each virus, four groups of 10 mice were inoculated intranasally with 3 LD50 of A/Wilson Smith/33 or A/Vietnam/1203/2004 virus in a 50 µl volume. At 24 h after inoculation, graded doses (10, 1, 0.1 mg/Kg) of PN-SIA49 or the control antibody (e137, 10 mg/Kg) were administrated to mice by intraperitoneal injection in a final volume of 0.2 ml. A subset of six mice in each group were weighed on the day of virus challenge and then observed and weighed every 2 days for 2 weeks after inoculation. Mice that lost more than 25% of their initial body weight were euthanized.
A subset of four animals treated with mAbs were euthanized on day 4 after inoculation, and whole lungs were homogenized in 1 ml of sterile PBS. Virus titers in lung tissue homogenates were determined by plaque titration in MDCK cell monolayer cultures.
Definition of the region bound by PN-SIA49 on HA
Hemagglutination (HI) assay
HI tests using mAb PN-SIA49 or e137 control antibody against live WS33 and VN04 viruses were performed according to standard protocols 
. Briefly, serial dilutions of purified mAbs in PBS were performed from initial concentration of 5 µg/mL. Positive and negative control ferret sera were diluted initially 1
10 in receptor-destroying enzyme from Vibrio cholerae (Denka Seiken, Tokyo). Serial dilutions of control sera or mAbs were pre-incubated with 4 HA units of virus per well. For WS33 virus, turkey red blood cells (RBCs) were added to a final concentration of 0.5%, whereas horse RBCs were used at a 1% suspension for VN04 virus. Normal ferret serum gave a value of less than 10. Specific HI activity of mAbs was calculated as the lowest concentration of mAb that displayed HI activity.
Protease susceptibility assay
Each reaction contained 3 µl of the anti-influenza vaccine season 2011–2012 (InflexalV-Crucell), which contains 30 ng of A/California/7/09 (H1N1) HA or 3 µl 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 37°C 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 37°C. Non-reducing SDS buffer was added to each reaction. Samples were boiled for ~2 minutes and loaded on a non-reducing 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 
, was used as primary antibody at 1 µg/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 (SuperSignal® West Pico Chemiluminescent Substrate, PIERCE) was added and incubated for 2 min.
To determine the pH required to convert the HA to the post-fusion form, pH titrations using the assay describing above was performed. Samples were exposed to a range of pH conditions (pH 4.5, 4.9, 5.3, 5.7, 6.1, 6.5, 7 and 8), neutralized and processed as described above.
Hemagglutinin cloning and mutagenesis
A/Puerto Rico/8/1934 (H1N1) hemagglutinin (A/PR/8/34-HA) was amplified as previously described 
using the following PCR primers:
The PCR products were cloned into the pcDNA 3.1D/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA). Subsequently, HA alanine mutants were generated using Gene Tailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA). A total of 20 A/PR/8/34-HA mutants were generated (His25Ala, His45Ala, Thr315Ala, Asn336Ala, Ile337Ala, Pro338Ala on the HA1 subunit and Trp357, Thr358, Gly359, Met360, Ile361, Asp362, Gly363, Trp364, Thr384, Ile388, Thr392, Val395, Asn396, Glu400 on the HA2 subunit. Sequence numbering refers to A/PR/8/34, GenBank accession number ABO21709).
Cytofluorimetric binding assays
The binding activity of PN-SIA49 was assayed using full-length wild type and mutants HAs. Human epithelial kidney HEK293T cells (ATCC CRL-1573) were transfected in 6 wells plate (Corning, Corning, NY, USA) (1×106 cells/well) with 4 µg of pcDNA 3.1D/V5-His-TOPO vector containing the HA nucleotide sequences described above. After centrifugation and fixation with 4% paraformaldehyde for 15 minutes at RT, the transfected cells were incubated for 30 minutes at room temperature with PN-SIA49 or conformational controls for H1N1 (RB62) at 10 µg/ml. Additionally, the isotype control, e137 (10 µg/ml) was introduced as well as untransfected cells and a mouse anti-influenza A HA (H1 subtype) monoclonal antibody (GeneTex Inc., Irvine, CA, USA) directed against a linear epitope to evaluate the transfection efficiency and the expression level for each HA. The cells were then washed with PBS and incubated for 30 minutes at room temperature with FITC-conjugated anti-human (Sigma Aldrich) or anti-mouse (Argene, Shirley, NY, USA) antibody. Afterwards, the cells were washed with PBS and analyzed by FACS. The FACS data were analyzed using the software Weasel w 2.5 (Waler+Eliza Hall, Institute of Medical Research, Parkville Victoria, Australia). The binding of PN-SIA49 to the different HA-mutants was then expressed as a binding percentage compared to wild-type. The data showing the PN-SIA 49 binding decrease between H1N1 wild type HA and H1N1 HA mutants, were obtained normalizing each PN-SIA 49 binding value to corresponding anti-H1 expression control values.
For the competition assay, serial dilutions of PN-SIA49 were used in combination with a fixed concentration (1 µg/ml) of mouse monoclonal antibody C179 (Takara Bio inc., Otsu, Shiga, Japan) which binds to an epitope on the HA stem region 
For sequences analysis the following software packages were used: SeqScape (Applied Biosystems), ClustalX (Toby Gibson), Bio Edit (Tom Hall, Ibis Therapeutics) and Treeview (GubuSoft). For molecular visualization and rendering UCSF Chimera package from the Resource for Biocomputing Visualization and Informatics at University of California, RasMol (Roger Sayle), Jmol (Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/
), Cn3D (United States National Library of Medicine, NLM) were used. Finally for data analysis and graphical editing GraphPad Prism was used.